Fun With Microbiology (What's Buggin' You?)

Capnocytophagia species

2011-12-17T23:36:00.014-05:00

Capnocytophagia species (C.ocracia) (Bacteria)

This isolate was cultured from the periodontal abscess of an otherwise health adult male.

On blood & chocolate agar, the 'aerobic' culture grew a light amount of mixed normal oral flora after 48 hours of incubation in 5% CO₂. The anaerobic culture also grew a light mix of normal oral flora with one exception. At 48 hours, a heavy growth of a small (~1-2mm), flat, tan coloured colony which had a mottled or bubbly texture and exhibited a spreading outer edge was noted

A gram stain showed this organism was a rather long, gram negative fusiform bacillus. First impressions from the gram stain and atmospheric requirement led me to believe this may be a Fusobacterium species often found as commensal anaerobic oral flora.

Aerotollerance Testing was performed subbing to Anaerobic Brucella Blood agar incubated anaerobically and to Chocolate agar placed in 5% CO_2.The anaerobic atmosphere contained 5% CO₂, 5% H₂ with the ballance N₂. After 24 hours of incubation weak growth was noted on the anaerobic plate while no growth was seen on the CO2 plate. After reincubation for an additional 24 hours the CO₂ atmosphere did support growth of the organism. It is interesting to note that Capnocytophagia was not isolated from the original aerobic culture although incubated in CO² in spite of being in greater numbers than the commensal aerobic oral flora which may, to some degree, compete for nutrients and surface area.

Initial presumptive identification using AN-Ident disks (Fisher Scientific.)¹ yielded little information because of the rather weak growth and the fact that the organism ultimately proved not to be an anaerobe. Enough growth was obtained to inoculate an API¹ Rapid 32A strip which after 4 hours incubation generated a profile code of 0713577707 identifying the isolate as Capanocytophagia species at 99.9%.

Capanocytophagia species is facultatively anaerobe as it is considered to be an aerobic organism that has the ability to survive and grow in anaerobic environments. Capnocytophagia appears to require an enhanced carbon dioxide atmosphere for optimal growth though some publications suggest that after several subcultures it can be encouraged to grow to some degree in air alone. It was interesting to note that our isolate grew first in the anaerobic atmosphere as I had also noted on an isolate I encountered several years ago. While both the Bactron IV Anaerobic Chamber and the CO2 incubatior had the same percentage of carbon dioxide, the difference in media, Brucella Blood Agar vs. Chocolate may account for the difference in growth.

The genus Capanocytophagia (previously known as DF-1 & DF-2 dysgonic fermenters) belongs to the family Flavobacteriaceae and currently consists of seven recognized species. Five species are clinically significant for humans; C.ocracea, C.sputigena, C.gingivalis, C.haemolytica, and C.granulosa. C.canimorsus is an oral commensal of dogs and the seventh species is C.cynodegmi also found in dogs and rarely in other species. As noted above the colonies are small reaching 4 – 5 mm after about 5 days of incubation. The size and colour is dependant on the atmosphere and media composition. Colour variation has been described as tan to yellow and even pink. The organism exhibits 'gliding' motility which can be seen as the spreading outer edge of the colony. This isolate was oxidase and catalase negative.

C.ochracea, C.sputigena, & C.gingivalis colonize the subgingival sulcus and other areas within the human oral cavity and are thought to play a role in the pathogenesis of localized juvenile periodontitis as well as other forms of peridontal disease. Bacteremia, endocarditis, conjuntivitis, keratitis and septic arthritis are the most common presentations in the immunocompromised host, particularly if they are granulocytopenic with leukemia or lymphoma as an underlying illness.

C.haemolytica and C.granulosa have been isolated from the supragingival plaque of health adults as from subgingival plaque in adult periodontitis.

Sensitivity Testing was not performed as there are no CLSI interpretive criteria for the species. Development of criteria is hampered by the slow growth and fastidious nature of this organism. Published reports suggest that Capnocytophagia are generally susceptivle to clindamycin, broad spectrum β-lactams, macrolides, cabapenems & fluroquinolones but are resistant to aminoglycosides. Results are variable for penicillin, expanded spectrum cephalosporins, vancomycin and metronidazole.

Capnocytophagia species on Brucella Blood Agar at 5 days Anaerobic Incubation.

(Click on photo to enlarge for better viewing)

Capnocytophagia species on Brucella Blood Agar at 72 Anaerobic Incubation.

(Note diffuse edge due to gliding motility)

(Click on photo to enlarge for better viewing)

Capnocytophagia species on Chocolate Agar after 72 hours incubation.

Note: Central raised colony with diffuse spreading edge (gliding motility)

(Click on photo to enlarge for better viewing)

Capnocytophagia species in gram stain of purulent material from periodontal abscess

(Click on photo to enlarge for better viewing)

Capnocytophagia species (arrows) in original gram (as above) along with other commensal oral flora, particularly Streptococcus in this photo.

(Click on photo to enlarge for better viewing)

Large clump of Capnocytophagia species as seen in gram stain made from a pick from colony growing on Anaerobic Brucella Blood Agar.

(Click on photo to enlarge for better viewing)

As above Photo; note length of gram negative (red) bacterial cells and their rather pointed ends.

(Click on photo to enlarge for better viewing)

Fisher AN-Ident Discs; Colistin, 10µg, Kanamycin, 1000µg, Vancomycin, 5µg were used.
BioMérieux Canada,Inc., St Laurent , QC, Canada

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Streptococcus pneumoniae

2011-11-22T22:25:00.018-05:00

Streptococcus pneumoniae (Bacteria)
Pneumococcal meningitis

Recently a 45 year old gentleman presented himself to the emergency department of our facility with suspected meningitis. The attending ER physician performed a lumbar puncture on the subject to obtain a sample of cerebral spinal fluid (CSF) which was noticeably cloudy. The specimen was sent to the laboratory where chemistry would measure the protein and glucose levels, haematology would perform a cell count and differential of the cells present, and to microbiology where we’d attempt to identify any infecting agent.

A number of different organisms can infect the blood stream (bacteraemia/septicaemia) and make their way to the meninges, the membranes that surround and protect both the brain and spinal cord.

The infection causes an inflammatory response and can produce various symptoms such as;

Headache

Stiff neck

Photophobia (dislike of bright lights)

Fever and chills

Drowsiness, confusion and possible delirium

Pneumococcal meningitis may not result in a rash which is common in Meningococcal meningitis (Neisseria meningitidis)

Pneumococcus can be found as commensal (normal) flora in up to 40% of the population and is the most common cause of meningitis in adults and in children over the age of two. The organism may reside in the back of the throat and in the nose where it can be passed on to others through close physical contact such as kissing or through coughs and sneezes. If the opportunity presents itself, this opportunist may invade the body to cause serious infections such as bacteraemia or meningitis. Still rather rare pneumococcal meningitis occurs at a rate of about 2 in every 100,000 of the Canadian population (1).

Predisposing factors for acquiring pneumococcal meningitis may include;

Diabetes

Compromised immune system

Pneumococcal pneumonia

Alcoholism

Head injury

Lacking a spleen

Other major chronic diseases

Laboratory Testing;
Chemistry - CSF is analyzed for it’s glucose and protein levels. Bacteria consume glucose for energy and a drop in the CSF to serum glucose ratio may provide a clue that a bacterium is the invading agent. Viruses do not utilize glucose for energy but may hijack the invaded cells ‘cellular mechanisms’ to produce protein necessary for its own reproduction an multiplication. Glucose levels that remain in the normal range with an increase of protein level may suggest a viral encephalitis. In our patient the levels were as follows;

In our patient;

Glucose = 2.3 mmol/L (2.2 - 3.9 mmol/L Normal Range)
Protein = 3.65 g/L (0.12 - 0.60 g/L Normal Range) -High

Haematology - Host cells present in the CSF are enumerated and differentiated which provide further clues to the nature of the illness. When an organism invades the body responds with an increase of white blood cells (WBC) to combat the invader. In other words, and increase in the number of WBC’s above the normal range suggests an infection. The type of WBC produced in response to the invader again provides further clues as to the invader. Neutrophils (=PolyMorphoNucleocytes=PMN) are the primary response to bacterial infections while viral agents may stimulate Lymphocytes.

In our patient;

Lymphocytes = 0.2 X 10^9/L (1.00 - 4.00 X 10^9/L Normal Range) -Low
Monocytes = 0.7 X 10^9/L (0.00 - 1.20 X 10^9/L Normal Range)
Neutrophils (PMN) = 17.2 X 10^9/L (2.00 - 7.50 X10^9/L) -High

Microbiology - In micro, we perform a gram stain on the specimen in an attempt to visualize the invading organism. A cytosine is usually performed which simply concentrates (via centrifugation) an aliquot of the CSF specimen onto a glass microscope slide for staining. In addition to concentrating the possibly small numbers of bacteria, the cytosine process ‘flattens’ the Neutrophils, thereby spreading them out and allowing better viewing of any bacterial cells present, particularly if ingested by the PMN.

The gram stain process not only makes the stained bacteria easier to visualize but may assist in differentiation of the invading bacterial species. Pneumococci (Streptococcus pneumoniae) are gram positive cocci and will appear as dark bluish-purple dots in pairs or short chains. Meningococcal (Neisseria meningitidis) are gram negative diplo-cocci and appear as red ‘coffee bean-like’ cells - in pairs with flattened adjacent sides. (gram negative bacilli may indicate a bacterial pathogen such as Haemophilus species while a gram positive bacillus may suggest Listeria species.)

In the case that presented to our lab, the CSF which is normally as clear as pure water, appeared as a cloudy or milky solution. Increased cellular immune response, the presence of bacterial cells and by-products may account for this altered appearance. Xanthachromia, a yellow colour due to the breakdown of red blood cells that may have entered the CSF was not observed. (This may be present in a Sub-arachnoid haemorrhage.)

The gram stain revealed gram positive cocci in pairs and short chains. Pneumococci often appear ‘lancet’ shaped which the adjacent pair of cells have a flattened common side while the opposite side is somewhat pointed or elongated.

Given the age of the patient and the gram stain result, pneumococcus was immediately suspected as the most likely culprit in this infection. A Quellung capsular swelling reaction was performed on the CSF to confirm our suspicions. I have described the Quellung Reaction elsewhere in this blog, and will only mention here that it is a specific and definitive test for pneumococcus. The Quellung reaction was observed allowing the lab to report a definitive diagnosis of pneumococcal meningitis to the doctor within an hour of the specimen being taken. Immediate treatment with appropriate antibiotics is imperative to prevent potentially catastrophic consequences.

Microbiological Results shown below;

Gram stain of Cytospin preparation of CSF (X1000)
Gram positive cocci in pairs and short chains suggestive of Streptococcus pneumoniae
Cystospin process has concentrated both the bacterial cells as well as the neutrophils (pink cells)
(click on photo to enlarge for better viewing)

Still at 1000X magnification but photo enlarged showing greater detail

Gram stain of Cytospin preparation of CSF showing greater detail. (X1000)
Insert on right may reveal evidence of capsule as clearing around bacterial cell.
(click on photo to enlarge for better viewing)

Quellung reaction in preparation of above CSF. (X1000 -Methyene Blue Counter Stain)
Clearing revealing presence of capsule viable around bacterial cells.
(click on photo to enlarge for better viewing)

Another view - ditto
(click on photo to enlarge for better viewing)

Quellung again at X1000 however I enlarged a portion of the photo to better illustrate the capsule surrounding the pneumococcal cells.
(click on photo to enlarge for better viewing)

Sheep Blood Agar Plate of CSF incubated overnight in 5% CO2 revealing alpha-hemolytic colonies (greening zones around colonies) typical of Streptococcus species. Colonies have a slight depression in the center typical of some Streptococcus pneumoniae strains. Insert shows colonial features in greater detail while purple arrow 'B' shows where colonies were tested for bile solubility (10% Sodium deoxycholate), another characteristic used as a confirmatory test for Streptococcus pneumoniae. Organism was sensitive to Optochin Taxo P Discs* (2) (not shown) adding further evidence of this bacterium being pneumococcus.
Phenotypic /biochemical identification was performed with the Phoenix Identification system (2) which also provided an antibiotic sensitivity summary. The patient would have been treated emperically when meningitis was first suspected or when the gram stain result (presumptive pneumococcal meningitis) result was sent STAT to to the attending ER physician.

(1) Meningitis Research Foundation of Canada
(2) BD™ Becton-Dickenson Diagnostics, Franklin Lakes, N.J. USA

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Neoscytalidium dimidiatum

2011-11-05T22:17:00.013-04:00

Neoscytalidium dimidiatum (formerly Scytalidium dimidiatum)

-Arthric synamorph of Nattrassia mangiferae
-Species, until recently known under the picnidial synamorph name of Hendersonula toruloidea occurs on a wide variety of tropical fruit trees, sometimes causing branch rot.

Macroscopic; Rapid growth quickly filling the plate, mature within three days. Effuse, hairy or wooly colonies which are white to greyish (or dark grey to blackish brown) with a cream colour to an ochraceous-yellow reverse. Melanin deficient mutants may occur.
Growth inhibited by cycloheximide therefore will not grow on selective media such as that used for dermatophyte isolation.

Microscopic; Branched and septate hyphae with no conidiophores. Arthroconidia (3-6 X 5-15 µm) develop and are 1-2 cells in length, flattened on the ends and may be rectangular, square, as well as oval to roundish becoming barrel shaped. The wide hyphae (6 -10 µm), and arthroconidia are brown (melanin) while the narrower side branches of the hyphae tend to produce pale arthroconidia. Melanin free variants (previously known as Scytadidium hyalinum) do occur, producing hyphae and arthroconidia that are invariably colourless but are identical to Neoscytalidium dimidiatum in every other way.

A picnidial form very occasionally develops in very old cultures and is currently considered to be Fusicoccum dimidiatum (formerly thought to be Nattrassia mangiferae. Picnidia, when present are large (100 - 300 µm). Picnidial conidia are hyaline when young with age but as they age may develop 1- 5 septa with a dark brown central region. Picnidia may be induced by prolonged growth on sterilized lemon or banana skins.

Pathogenicity; Known to cause nail and skin infections with rare reports of deep-seated infections such as brain infections, sinusitis, lymphadenitis and endophthalmitis with higher incidence of fungemia in immunocompromised patients. Infection is primarily found in patients from tropical to sub-tropical regions.

Neoscytalidium dimidiatum on SAB agar after 3 days incubation.
(click on photo to enlarge for better viewing)

Neoscytalidium dimidiatum on SAB agar Reverse & Surface after 5 days growth.
Surface shows impression where hyphae were removed in celluloid tape examination.
(click on photo to enlarge for better viewing)

Hyphae 40X LPCB

Broad Septate Branching Hyphae (6 -10 µm)
Note narrower, un-pigmented side branches.
(click on photo to enlarge for better viewing)

Dark (melanin), Broad, Septate, Branching Hyphae (6 -10 µm)
Arthroconidia present. (X400)
(click on photo to enlarge for better viewing)

Broad septate hyphae and contiguous arthroconidia (LPCB X 400)
(click on photo to enlarge for better viewing)

Ditto
(click on photo to enlarge for better viewing)

Pigmented arthroconidia as contiguous cells.
(click on photo to enlarge for better viewing)

Arthroconidia (LPCB X600)
(click on photo to enlarge for better viewing)

Broad septate hyphae and two celled conidia.
Unstained preparation from Corn Meal Agar (magnification not noted)
(click on photo to enlarge for better viewing)

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Slide Culture Technique

2011-11-04T23:44:00.019-04:00

Preparation of a slide culture can be useful in the study and identification of an unknown fungal isolate. While the 'sticky tape' preparation is quick and easy to perform, it can be disruptive, with salient features altered or destroyed. The slide culture when performed properly is more gentle allowing for the examination of intact structures & features as they naturally developed. The technique is quite easy to perform and I have outlined the basic steps in the photos that follow.

(1) Start by getting a plate of fungal media, Saboraud-Dextrose as shown above, and some means of cutting the agar. Pictured here is a sterile scalpel however they can be relatively pricey for such a small job. I prefer to use a microscope cover slip as the edge is thin, sharp and straight. A 20 X 20 mm cover slip can be used however the 20 X 40 cover slip allows for batter grip. Simply plunge or drag the edge of a cover slip into the agar surface, cutting out small blocks of agar somewhat about 1/2 to 3/4 of an inch square. as shown below.
(some people have used the open end of a sterile test tube as a punch to cut out a round plug of agar for the same purpose)

(2) Above you can see how I sliced the agar into squares that would be slightly smaller than a 20 X 20 mm glass microscope cover slip, using a glass cover slip as a knife. Cut as many squares as you require for the current and near future projects.

(3) A row of squares cut into the agar is seen above. Cut as many as you need for your project and the remaining plate can be placed into a plastic bag and refrigerated for future projects. (note the expiry date of the agar)

(4) Remove an agar square that you have cut into the plate using the same cutting tool (scalpel or cover slip) that you used to previously.

(5) Place the agar block onto a clean glass microscope slide. You can place one per slide or two per slide as I sometimes do. You can also place two different agars on the same slide. (eg. SAB and Corn Meal)

(6) The slide can then be placed in a clean petrie dish which will prevent contamination and preserve moisture during incubation. I like to raise the slide off the bottom of the petrie dish. Moisture in the between the plastic surface of the dish and the glass slide itself might create enough surface tension between the two making removal of the glass slide difficult without disrupting the delicate growth. You can use plastic or wooden stir sticks for this purpose. Some people sacrifice an uninoculated media plate such as blood agar and place the slide directly onto the agar surface. The agar acts as a source moisture during incubation however the surface tension may make remove of the slide somewhat more difficult.

(7) Using a sterile instrument (loop, needle, etc), transfer some of the fungus (spores, conidia etc) from the specimen being cultured to each of the four sides of the agar block. Just a quick touch of the isolate to the four edges as shown below.

(8) Transfer the fungus to the agar block's sides. It isn't necessary to smear the entire side but rather just touch the center of the agar block.

(9) After inoculation, place a clean cover slip on the surface of the agar block. (Remember to do this as it is easy to forget!!).

(10) A few drops of sterile water can be added to the petrie dish as an additional source of moisture which may be beneficial to slow growing fungi which may dry out with prolonged incubation. In general this is not necessary as the dish will be partially sealed up.

(11) The plate is now partially sealed with Parafilm™ or a bit of cellulose tape. If fully sealed the plate may fog up and moisture condense on specimen. The tape partially seals the plate, still allowing it to breath. The sealing of the plate also minimizes contamination of both the culture and the environment.

The slide culture is now ready for incubation. Incubate the slide at an appropriate temperature (room temperature to 30C for most fungi) and for an appropriate length of time. Fast growing fungi can overgrow the agar block very quickly so monitor the growth visually daily. One may wish to make several side cultures of the same fungus and examine one per day to observe how the structures develop.

To examine the slide culture, remove the slide from the petrie dish and then gently remove the cover slip from the agar block using plastic forceps or gloved fingers. The fungus should have adhered to some extent to the glass cover slip. Place a drop of LPCB* onto a clean microscope slide and then place the cover slip from the slide culture (growth down) onto the LPCB. The slide is now ready for examination under the light microscope. Don't throw the agar block away as if you remove it you will find that the fungus also adhered to the microscope slide. Just add LPCB and cover slip the growth. You get two cultures from one block - top and bottom.

*LPCB: Lacto Phenol Cotton Blue stain.

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Curvularia sp.

2011-05-09T22:42:00.012-04:00

Curvularia species.

Filamentous fungus; Hyphomycetes, Dematiaceae, Genus: Curvularia

Ecology;
There are numerous species of Curvularia, many of which are facultative pathogens of tropical or subtropical plants, but some species can be isolated in temperate agricultural areas. They are particularly common as saprobes or weak pathogens on grasses.

Macroscopic;

Rapid growth

Woolly texture

Whitish colonies becoming olive brown on the surface and reverse.

Microscopic;

Septate hyphae, generally brown in appearance

Conidiophores brown, erect, simple or branched. geniculate (1), producing conidia in sympodial (2) order.

Poroconidia (3), ellipsoidal, often gently curved (hence the name), brown in colour, usually with 3 to 4 true septa. Flat conidial scar on release.

(Poro)conidia have transverse septa, ie. wall to wall and not distoseptate (4) as with Bipolaris species or Drechslera species.

Curvularia hyphae, Conidophores and Conida (LPCB X250)
(Click on photo to enlarge for better viewing)

Curvularia - Multi-celled mature brown poroconidia (LPCB X400)
(Click on photo to enlarge for better viewing)

Curvularia - conidiophore bearing curved, brown, multicelled conidia
(click on photo to enlarge for better viewing)

(Center) Conidiophore with numerous conidia attached (LPCB X400)
Note: 100 µm bar in upper right
(Click on photo to enlarge for better viewing)

3 Young conida attached at end of conidiophore (LPCB X400)
(Click on photo to enlarge for better viewing)

Geniculate (bent) conidiophore bearing multicelled conidia (LPCB X400)
(Click on photo to enlarge for better viewing)

Curvularia conidiophore with geniculate (bent) apical end bearing sympodially placed, curved, multicelled poroconidia (LPCB X400)
(Click on photo to enlarge for better viewing)

Ditto - Geniculate (bent) distal end of conidiophore with still attached curved poroconidia
(Click on photo to enlarge for better viewing)

Pathogenicity;
Not tremendously pathogenic, however, may be found in immunocompromised patients with increasing frequency. Reports of infection following traumatic injury. Occasionally found causing onychomycosis, keratitis, sinusitis, mycetoma, pneumonia, endocarditic, cerebral abscesses and disseminated infection. Mycetomas are most frequently found in animals.

(1) Geniculate - bent abruptly, as in a bent knee.
(2) Sympodial - continued, successive apical growth with each subsequent point of growth next to the previous one and terminating at somewhat higher level.
(3) Poroconidia - conidia produced through a pore in the conidiophores.
(4) Distoseptate - conidia subdivided by inner wall layer only. A thin septum lacking a septal plate and penetrated by cytoplasm junctions, possibly formed by invagination from the lateral wall.

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Trichomonas hominis

2011-04-23T17:07:00.004-04:00

Trichomonas hominis

Parasite (Flagellate)

A relatively common flagellate which may be overlooked or unrecognized during examination of fecal specimens for parasites. This organism is found worldwide in both warm and temperate climates. Its detection in fecal specimens does not seem to be related to gastrointestinal illness although it is often recovered from diarrheic stools. Trichomonas hominis is considered to be non-pathogenic.

As with Trichomonas vaginalis, there is no known cyst stage therefore it must be acquired by ingesting the trophozoite directly. The troph probably survives the acid stomach environment by ingestion along with some protective substance such as milk. After passing through the stomach, they tend to take up residence in the cecal region of the large intestine where the feed on bacteria.

Trophozoites are about 5 to 15 µm by 7 to 10 µm in size and are roughly pyriform in shape. As with Trichomonas vaginalis, they have both an axostyle and undulating membrane, which helps to differentiate them from other fecal parasites that may be present. The undulating membrane extends the entire length of the body, in contrast to that of Trichomonas vaginalis.

Diagnosis; In freshly passed stool they may be recognized by motility provided by the characteristic movement of flagella and rhythmic beating of the undulating membrane. These rather small organisms are difficult to see in the fresh specimen and are easily overlooked in stained preparations.

Both Trichomonas hominis and Trichomonas vaginalis are site specific in that neither can survive in the other’s environment. Attempts to transplant T.hominis into the vaginal were unsuccessful.

While prevalent worldwide, it is recovered with greater frequency in warmer climates and from children more than in adults. Prevention is by interrupting transmission which is accomplished though increased hygiene and improved sanitary conditions. Treatment is not warranted.

Trichomonas hominis in stool specimen (Iron-hematoxylin stain x1000)
(Click on image to enlarge for better viewing - 1024 X 768 px)

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Cunninghamella species

2011-04-19T13:49:00.010-04:00

Cunninghamella species
-filamentous fungus in the order of Mucorales.

Ecology;
Widespread distribution primarily in sub-tropical climates - commonly found in soil, animal matter and soil. Has been isolated from cheese.

Macroscopic;
Extremely fast growing fungus producing wooly mycelia which can fill a petrie dish in 2 to 3 days. Mature matt-like surface growth appears white to grey, darkening with age. Reverse is white/pale/buff.

Cunninghamella on Sabouraud-Dextrose Agar after 48 hours incubation at 30C

Microscopic;

Hyphae are broad and aseptate or rare septations

Sporangia are long, branched and ending in swollen vesicles about 40 µm in diameter. Vesicles on lateral branches are generally smaller.

Vesicles are covered with spine-like denticles, each supporting a single round to oval sporangiolum (7 µm to 12 µm).

Each sporangiolum contains one spore which can be smooth walled or finely echinulate (ie. is spiny or has small prickles)

Zygospores may be present (spherical, 25-55 µm diameter, brownish with tuberculate projections. Heterothalic.

Rhyzoids may be seen.

Cunninghamella sporangiophore covered with sporangiola

Cunninghamella species sporangiophore bearing sporangiola attached to surface by a denticle.
(Click on photo to enlarge for better viewing)

Cunninghamella sporaniophore with terminal vesicle. Sporangiophores dispersed revealing the denticles (arrow) by which they were attached to the vesicle. (LPCB X400)
(click on photo to enlarge for better viewing)

Cunninghamella sporangiospores
(Click on photo to enlarge for better viewing)

Physiology;
A thermophilic species with good growth at 30C as well as at 45C. Growth is inhibited by cyclohexamide.

Pathogenicity;
An opportunist. Cunninghamella is agent of zygomycosis known to have cause pneumonia in immunocompromised children, disseminated disease in renal transplant recipients and infected other severely debilitated patients such as those with AIDS. C.bertholletiae is considered to be the only pathogenic species. It can be distinguished from the non-pathogenic C.elegans by its ability to grow at 40-45C.

Cunninghamella computer wallpaper (1024 X 768) as posted

(Click on photo to enlarge for better viewing)

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Syncephalastrum species

2011-04-19T11:59:00.012-04:00

Syncephalastrum species
-A filamentous fungus classified within the order Mucorales.

Ecology;
Syncephalastrum species are saprobes commonly isolated from animal dung and soil primarily in tropical or subtropical regions.

Macroscopic;
Very rapid growth - mature within 3 days. Abundant aerial hyphae gives a wooly or cotton candy-like texture. Pale to dark grey shades on the surface with a white or pale reverse.

Syncephalastrum on Sabouraud's Dextrose Agar at 72 hrs at 30C

Microscopic;

Broad hyphae -septa absent or rare.

Sporangiophores are 10-25 µm wide, often branched, having a terminal vesicle up to 80 µm in diameter, which produce finger shaped merosporangia (4-6 µm by 9-35 µm) over the entire surface.

Sporangiospores (merospores) are round and formed in a linear series, between 3 to 18 in number within in the interior of the merosporangia. They are smooth in texture, pale brown, spherical to oval, ~3-7 µm in diameter.

Rhyzoids are usually present

Sporangiophore arising from hyphae with terminal vesicle with merosporangia radiating out from the surface. (LPCB X400)
(click on photo to enlarge for better viewing)

Mature Syncephalastrum sporangiophores (LPCB x400)
(click on photo to enlarge for better viewing)

Ditto
(click on photo to enlarge for better viewing)

Syncephalastrum sporangiophore with radiating finger-like merosorangia containing individual merospores.
(click on photo to enlarge for better viewing)

Finger-like merosporangia free of sporangiophore, each containing merospores
(click on photo to enlarge for better viewing)

Pathogenicity;
Considered to be non-pathogenic to humans although a case of cutaneous infection has been reported (Kalama & Thambiah 1980). May be found as a contaminant.

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Mucor Species

2011-04-03T14:53:00.009-04:00

Mucor species
Fungus

The genus Mucor is a filamentous fungus with several species, few of which grow well at 37C (body temp.) and thereby able to infect humans. Mucor species have been implicated as an agent in zygomycosis, particularly in the debilitated patient. Mucor is ubiquitous in nature, found in the soil or growing on decaying vegetative matter.

Macroscopic Appearance (Colony) ~25-30C;
Mucor is a rapidly growing fungus which will fill a culture plate in a matter of a few days with a woolly growth resembling cotton candy. New growth is white in colour but turns a greyish-brown with aging. The reverse remains a pale white.

Mucor On SAB Agar Plate 48hrs at 30C

Microscopic Morphology;
Mucor has broad hyphae which are scarcely or non-septate. Sporangiophores are long, may be branched and terminate in a round spore-filled sporangia (50µm-300µm diameter). The sporangia has a thin wall which when mature dissolves (or is disrupted) to release round or somewhat ellipsoidal sporangiospores (4µm-8µm diameter). With the spores scattered, the collumella which bore the sporangia is visible, sometimes leaving a collerette at the base of the sporangium.

Welcome to the Jungle
Sparsely septated hyphae, sporangiophores bearing sporangia (LPCB X100)
(click on photo to enlarge for better viewing)

Mucor sporangiophore bearing dematiaceous sporangia containing sporangiospores (400X)
(click on photo to enlarge for better viewing)

With spores dispersed, collumella is revealed with collarette remaining beneath (arrows)
(click on photo to enlarge for better viewing)

Mucor species are capable of producing chlamydospores.
Mucor differs from Rhizopus species in that it fails to produce rhizoids and from Absidia species by the absence of apophysis beneath the sporangium.
Mucor is inhibited by cyclohexamide.

Treatment;
Amphoteracin B, Ketoconazole, Itraconazole and Voriconazole have shown to have activity against Mucor, however clinical response may be variable.

Mucor species computer screen wallpaper (1025 X 768) when posted.
(click on image to enlarge for better viewing)

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Hookworm & Trichostrongylus Ova

2011-04-02T11:49:00.013-04:00

Hookworms & Trichostrongylus sp.
-Nematodes (Roundworms)

Hookworms;
Ancycostoma duodenale (old world hookworm) -predominant in South-East Asia, China, India, Northern Africa and Southern Europe.
Necator americanus (new world hookworm) - America, Caribbean, Central America, Southern Africa, Polynesia -virtually World-wide.

Old & New World labels are historical and somewhat of a misnomer as geographic distribution overlap.
Hookworms are found in warm, moist areas of the world.

Adult Worms;
Adult males are about 7-11 mm long with A.duodenale usually slightly larger than N.americanus. Adult worms are rarely seen as they usually remain firmly attached to the intestinal mucosa by their mouth. A.duodenale has well developed teeth while N.americanus has cutting plates facilitating attachment. As such, diagnosis is usually made by finding hookworm eggs in stool specimens. Eggs (ova) are essentially identical for both hookworm species.

Infection is acquired through skin penetration of the filariform larvae from soil. Hookworms have hyaluronidase activity which facilitates the infective larvae penetration through skin. After penetration, the larvae are carried to the heart and then to the lungs where they enter the alveoli and migrate through the bronchi to the trachea and pharynx. Once they reach the pharynx they are swallowed and travel to the small intestine where they attach to the mucosal surface and further mature. Females usually begin to deposit eggs about 5 months after initial infection.

Eggs (~56µm to 75µm X 36µm to 40µm) that are passed in the stool are usually in the early cleavage stage and appear rather oval with broadly rounded ends and a clear space between the embryo and egg shell. Eggs will hatch within 1 to 2 days when in moist, shady warm soil. Infective filariform larvae develop within 5 to 8 days of hatching under optimal conditions and can remain viable in the soil for several weeks.

Hookworm Egg - Note broadly rounded ends, clear space around developing cells.

(click on image to enlarge for better viewing)
All photos from formalin-ethyl acetate concentrate at 400X

Clinical Disease;
Hookworm infections cause more morbidity rather than mortality. Symptoms usually are related to the larval load (how many). Infections may go unnoticed or may cause pruritus with further complication by secondary infection through scratching. Depending on the number of migrating larvae, pneumonitis may result.
Once attached to the intestinal mucosa, symptoms may include;

Necrosis of intestinal tissue

Blood loss through ingestion by the worm or direct bleeding facilitated by an anticoagulant secreted by the worm

Fatigue

Nausea & Vomiting

Abdominal pain

Diarrhea with stools that may be tinted red to black due to blood loss.

Chronic infections may result in anaemia from the blood loss.

Again, diagnosis is made by finding the hookworm eggs in stool samples, either in direct, concentrate or stained smears. Results should be reported as “Hookworm Ova” present as species (N.americanus, A.duodenale) cannot be determined by the egg.

Prevention & Treatment;
Sanitary treatment & disposal is the most effective way of reducing exposure to the filariform soil larvae. Wearing of shoes may also help prevent infection.
Anti-helminthic drugs such as albendazole prove effective in treatment.

Just for comparison- A Hookworm egg in the upper right compared to an Ascaris egg in the lower left of photomicrograph. Fecal concentrate at X250)
(click on photo to enlarge for better viewing)

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Trichostrongylus Species; (several species can infect humans with varying severity)
This nematode (roundworm) has worldwide distribution and is commonly found in herbivores.
Trichostrongylus species are similar to hookworms as they also take up residence on the mucosa of the small intestine however they differ in not having teeth or cutting plates.

Infections is usually acquired by ingestion of the infective larvae on contaminated plant material. Trichostrongylus larvae reach the small intestine without any migratory pathway through the lungs and mature there within 3 to 4 weeks.

The Trichostrongylus ova appear similar to Hookworm ova however they are slightly larger (~73µm to 95µm X 40µm to 50µm) and tend to have more pointed ends.
Eggs deposited in warm, moist soil may hatch as quickly as 24 hours and develop into infective larvae after about 60 hours.

Trichostrongylus Egg - Note similarity to Hookworm Egg above however end more pointed.
(click on image to enlarge for better viewing)

Trichostrongylus Egg - Note much more pointed end than Hookworm
(click on image to enlarge for better viewing)

As with the Hookworms, symptoms from Trichostrongylus infections are related to the worm burden and subsequent damage to the intestinal mucosa. Haemorrhage and desquamation may occur as with Hookworm infections.
Symptoms are not usually clinically significant unless there are large numbers of worms present.

Diagnosis is usually made through egg identification in the stool.

Prevention and Treatment;
Herbivores constantly reinfect grazing areas therefore the only effective prevention is the proper cleaning and cooking of vegetable foodstuffs. Treatment again is with anti-helmithic drugs such as albendazole.

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Entamoeba histolytica/dispar

2011-03-31T15:47:00.008-04:00

Entamoeba histolytica/dispar

Intestinal Protozoa: Amoebae

The pathogenic Entamoeba histolytica and non-pathogenic Entamoeba dispar are indistinguishable in stained smears and must be reported as E.histolytica/dispar until speciation can be confirmed by serologic or antigenic means. For simplicity I’ll refer to this complex throughout this text as E.histolytica with the assumption that species confirmation will follow.

Distribution;
Entamoeba histolytica has worldwide distribution however is more prevalent in the tropics and sub-tropics than in colder climates. As much as 10% of the worlds population may be infected with E.histolytica and ranks only behind malaria and Schistosomiasis as a cause of worldwide mortality.

Yet with this relatively large percentage of worldwide infection, only a smaller number of infected go on to develop severe dysentery or extra-intestinal disease. Over the years, a number of theories have been proposed for this discrepancy however, recently is has been shown that two morphologically identical species exist. Isoenzyme patterns for enzymes such as hexokinase and phosphoglucomutase differ between invasive and non-invasive species and can be used to confirm identification. Monoclonal antibodies and DNA probes have also been developed for this purpose.

Poor sanitation and overcrowding are major contributors to the prevalence and transmission of E.histolytica. Infection is usually acquired by ingestion of the cyst through contaminated water or food. After ingestion, the cyst passes through the acidic environment of the stomach to the more alkaline intestine. The excystation produces trophozoites which then through enzymatic and contact dependant cytotoxic means are able to attach and penetrate the intestinal mucosal wall A small crater-like lesion develops and progresses to a classic flask shaped ulcer as the organisms establishes itself. Cysteine proteinases secreted by the trophozoite degrade collagen, elastin and fibronectin. Phospholipases and haemolysins may also facilitate spread through the tissues. A pore-forming protein is the final mechanism in cellular death as it allows the influx of calcium ions into the cell.
Cell mediated immune response has little effect against E.histolytica and the organisms may be able to suppress immune response to some degree.

Clinical Symptoms;
Manifestations of the disease can present itself in three ways;

Asymptomatic cyst passer - does not exhibit illness but is a source of cysts for infecting others.

Acute amoebic colitis or dysentery - patients have fever, cramping and abdominal pain resulting in numerous bloody stools per day. Ulceration may result in perforation of the bowel and peritonitis. As trophozoites do not survive well outside of the body, these individuals are not a significant source for infecting others.

Invasive disease - development of the classic flask shaped ulcer may result in perforation and distribution of the trophozoite through portal circulation. The liver is the primary organ colonized by E.histolytica extra-intestinal trophozoites however pluropulmonary and central nervous system infections have been described.

Diagnosis;
Laboratory diagnosis is made through identification of the trophozoite or cysts in the stool. A minimum of 3 stools over a period of 10 days is recommended. Liquid stools are associated with acute amoebas where trophozoites are the form of E.histolytica present. Such stools should be examined immediately (~30 min) for motile trophozoites. If present, trophs may show a directional progressive motility as pseudopod develops in the direction of movement.
Cysts are usually found in semi-formed or formed stools.

Stools, whether liquid or formed should be preserved in PVA or SAF (1) preservative to prevent degradation. Preserved samples can be used to make concentrations and permanent stained slides for microscopic examination.

Morphology;
Trophozoites; Trophozoites range in size from 10µm to 60µm . Trophozoites of E.histolytica may on occasion be seen to have ingested red blood cells within the cytoplasm, more commonly in the acute phase of illness. Cytoplasm usually is not vacuolated and has a fine or even consistency. The nucleus is characteristic and best examined in the stained preparation. There is a small, centrally located karyosome with fine and evenly distributed peripheral chromatin on the nuclear membrane.

E.histolytica trophozoite; Iron Hematoxylin Stain (1000X)

SAF preserved concentrate of stool specimen showing E.histolytica trophozoite (400X)

Cysts; E.histolytica cysts are about 10µm to 20µm in diameter and have up to 4 nuclei. Chromatoidal bars, if present are ‘cigar’ shaped in that they have rounded ends with parallel sides.

E.histolytica cyst showing 4 nuclei an chromatoidal bars with rounded ends.
(click on photo to enlarge for better viewing)

E.histolytica cyst with 4 nuclei and chromatoidal bar
(click on photo to enlarge for better viewing)

Treatment;
Most patients infected with E.dispar clear the organisms spontaneously within 8 to 12 months.
Metronidazole is effective in eradicating E.histolytica.

(1) Preservatives - Poly Vinyl Acetate (PVA) & Sodium Acetate Acetic Acid Formalin (SAF)

E.histolytica trophozoite: Intended as computer wallpaper (1024 X768) when posted
(click on photo to enlarge for better viewing)

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Phialophora varrucosa

2011-01-31T21:22:00.011-05:00

Phialophora varrucosa
Fungus (Black Mould)

One of the agents responsible for chromoblastomycosis, a slow moving fungal infection of the skin and subcutaneous tissue, usually initiated a splinter or thorn. A cosmopolitan saprobe commonly isolated from soil and decomposing wood. This infection is more common in tropical or sub-tropical climates where it can difficult to treat but is rarely fatal.
Other agents causing chromoblastomycosis are;
-Fonseca pedrosoi
-Cladosporum carrionii
-Fonseca compacta

Growth;
Exhibits slow to moderate growth, usually maturing in about 7 – 12 Days.

Macroscopic Appearance;
Colonies are woolly to velvety, dark grey, brown or olivacious black on the surface and reverse.

SAB agar at 10 Days incubation
(Click on any photo that follows to enlarge for better viewing)

Microscopic Appearance;
Dematiacious (melanin pigment) - Hyaline to brown, septate hyphae.
Phialides are pale brown to brown, bottle or vase or shaped with a darker collarette at the apical end. Phaialides are located laterally or terminally on the hyphae.
Conidia are unicellular, smooth and thin walled, hyaline to brown and round or ovoid (1-3 X 2-4 µm) which accumulate at the apex of the collarette giving the appearance of a vase of flowers.

In chromoblastomycosis (histologic specimen), the organisms appear as dark round cells (copper pennies) 5 – 12 µm dia.

Phialophora verrucosa is distinguished from other fungi by its unique phialides with their collarettes. The shape of the collarettes themselves distinguishes Phialophora verrucosa from Phialophora richardssiae, the former being cup shaped, the latter having a flatter, saucer shape.

Vase shaped phialides with round to oval conida at apex. Dark brown (containing melanin) cup shaped collarette at apex. Hyaline to brown (dematiacious ie. containing melanin), septate hyphae,

Ditto

(Click on any photo to enlarge for better viewing)

Vase or bottle shaped phialide showing dark collarette with oval conidia

Voriconazole, Itraconazole with 5-flucytosine have has shown to be effective in treating this difficult infection. Cryosurgery has also been employed.

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Pseudallescheria boydii/Scedosporium apiospermum

2011-01-17T23:42:00.015-05:00

Pseudallescheria boydii/Scedosporium apiospermum
Fungus
Pseudallescheria boydii (sexual state) -Teliomorph
Scedosporium apiospermum (asexual state)

The names above refer to the same fungus exhibiting different reproductive states. The sexual state name (Pseudallescheria boydii) takes priority over the asexual state name (Scedosporium apiospermum). However, the sexual state often is not present in clinical specimens and unless it is observed, the fungus must be referred to by the asexual name of Scedosporium apiospermum.

Ecology;
The fungus is cosmopolitan and can be isolated from rural soils, polluted water, composts and manure.

Macroscopic (Colony);

Moderately rapid growing colony at 25C which matures in about a week.

Texture is woolly to cottony.

Colony is white becoming greyish or smokey-brown on the surface; reverse is pale with brownish-black zones.

Sexual state P. boydii is inhibited by cycloheximide (fungal selection agars) while the asexual state S.apiospermum is not inhibited.

Microscopic;

Hyphae are hyaline, septate (2 - 4 µm dia.)

Conidiophores bearing annellides are of varying length and exhibit little differentiation from the vegetative hyphae.

Conidia (annelloconidia) are unicellular, pale brown, ovoid with truncate bases formed singly or in small clusters at the ends of the conidiophores or from short annellidic necks arising directly from the hyphae.

Fascicles of conidiophores bound together in synemata are sometimes present and is a variation of the asexual state referred to as the Graphium synanamorph (one anamorph somewhat resembles a wheat sheaf, the other anamorph a bulb on a stalk[ not flattend as in LPCB prep.]).

Large (50 to 250 µm) brown cleistiothecia (of the sexual P.boydii) may develop after 2 to 3 weeks of incubation. More likely to be found at center of colony.

Ascospores are yellow-brown and ellipsoidal in shape.

Scedosporium apiospermum hyphae & (annello)conidia
(LPCB sticky tape preparation at 250X)
-click to enlarge-

Scedosporium apiospermum septate hyphae, conidiophores & conidia
(LPCB sticky tape preparation at 400X)
-click to enlarge-

Annelloconidia showing truncate base/scar -wider at apex
LPCB 500X
-click to enlarge-

Scedosporium apiospermum asexual Graphium synanamporph
(Fascicles of conidiophores bound together in synemata -wheat sheaf appearance)
-click to enlarge-

Graphium synanamorph
(Slide culture on Corn Meal Agar-edge of agar at photo bottom 250X)
-click to enlarge-

As Above Description
-click to enlarge-

Immature Cleistothecium at 10 days Corn Meal slide culture LPCB
(Hyphae & Conidia can be seen in surrounding background)
-click to enlarge-

Immature Cleistiothecium of Pseudallescheria boydii
(Corn meal agar slide culture-unstained X400 ~10 days)
-click to enlarge for better viewing-

**New: Feb 17th, 2011

Split Mature P.boydi clestiothecium
(taken with the DMD-108 microscope)
(click on image to enlarge)

Mature P.boydi clestiothecium split and releasing ascospores
(ascospore inset)
(Click on photo to enlarge for better viewing)

P.boydi clestiothecium split open revealing lemon shaped ascospores within.
(click on photo to enlarge for better viewing)

***Note: The sexual stage of P.boydi was induced using corn meal agar both as a slide culture and extended incubation of the plate media. The last two photos (Feb 17th) from corn meal agar media after about 8 weeks of incubation. A small segment of agar was cut out of the plate and placed on a glass microscope slide. A second slide was placed on top of the agar and the agar was crushed between the two (The clestiothecia of P.boydi were much more difficult to split open than those of Aspergillus nidulans.) The the two slides were then separated, leaving material on both. A drop of KOH was added to help clear and define the organism.

Pathogenicity;
An occasional agent of infections including mycetoma, cutaneous or subcutaneous invasion, most often of the feet and hands. Has been implicated in otitis, sinusitis, keratitis, endophthamitis, pneumonia, endocarditic, meningitis, osteomyelitis and cerebral abscesses. Disseminated disease more likely to occur in debilitated patients or those immunocompromised. Is also now recognized as an agent of phaeaohyphomycosis

Susceptibility;
Reference

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Cyclospora cayetanensis

2011-01-09T20:41:00.006-05:00

Cyclospora cayetanensis
(Cyanobacterium-Like Body/Coccidian-Like Body/CLB)
Coccidian Protozoan Parasite

Though the genera Cyclospora has been known about for some time as pathogen of vertebrates, this nasty little species began to be increasingly reported in humans in the late 1970’s. First thought to be associated with blue-green algae, it was referred to as a Cyanobacterium-Like body. Others thought it was a larger variant of Cryptpsporidum. The organism was finally identified as belonging to the genus Cyclospora based on sporulation studies which revealed that each oocyst contains two sporocysts which in turn contain two sporozoites.

Cyclospora can be found worldwide however it may exhibit greater prevalence in rainy areas where outbreaks coincide with the rainy season. Infection is primarily acquired by ingestion of water bearing the oocyst or produce which has been irrigated with contaminated water. There is no proof of person to person infection as the oocyst needs about 10 days outside of the body to mature and become infective.

On ingestion, the oocyst takes up residence within cells (enterocytes) of the small intestine. Cyclospora appears to have both asexual and sexual reproduction with the infective oocyst being the product of sexual reproduction. The invasion of the intestinal enterocytes is thought to stimulate cytokine production which causes inflammation, electrolyte secretion and ultimately osmotic diarrhoea.

Otherwise healthy individuals may suffer a prolonged, yet self-limiting watery diarrhoea. Symptoms may include vomiting, nausea, fatigue, anorexia, fever, cramping and weight loss. Episodes of diarrhoea may alternate with constipation. Symptoms may begin 2 to 10 days after exposure and may last up to 7 weeks.
In immunocompromised patients such as those with AIDs, the illness can persist longer and have devastating consequences.

Diagnosis and Identification;
The organism is shed irregularly so stool specimens should be collected periodically if Cyclspora is suspected. The organism can be identified by several means including;

Wet-preps of formalin-ether concentrates - the spherical organisms are about 8 to 12 µm and appear refractile in unstained preparations.

Cyclospora exhibits variable acid-fast properties and organisms will stain to various degrees from virtually unstained through increasingly intense shades of pink. Stained oocyst may appear slightly smaller than the unstained 8 to 12 µm due to shrinkage. Cells often display a folded or wrinkled appearance. Cyclospora does not stain well with the Trichrome method.

Cyclospora will auto fluoresce under ultra-violet light, appearing greenish under 450 nm wavelength and bluish under 365 nm.

Treatment;
Septra (Trimethoprim-Sulfamethoxazole) is commonly used in treatment.

Note: Cyclospora have been known to survive routine chlorination treatment.

(click on photo to enlarge for better viewing)

Above: Cyclspora cayetanensis as stained by the modified Iron-Hematoxylin staining method.
Organism exhibits variable acid-fastness thereby appearing clear/unstained right through various intensities of pink. Note too the wrinkled or folded appearance the organism can have in the stained preparation. Wrinkling and a slightly smaller diameter may be due to shrinkage during the staining process. (photos X1000 oil immersion)

(click on photo to enlarge for better viewing)

Formalin-ether stool concentrate wet preparation showing unstained Cyclospora cayetanensis.

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Streptococcus anginosus group

2010-12-26T17:12:00.014-05:00

Scanning a gram stain of a body fluid, you come across what appears to be a jumble of purple (gram positive) dots within a white blood cell. At first glance the dots do not appear to be arranged in clusters as would be expected if Staphylococci, nor do they appear to be very convincing chains of cells as Streptococci would exhibit. Focusing up and down through the depth of material on the slide, no clear ghost cells bearing sporadic beading of crystal violet (purple-blue) stain as might be expected with mycobacterium is evident. (See previous post on Mycobacterium tuberculosis).

What the heck is this? An organism?…an artifact of staining?

A colleague encountered this situation however, with the specimen site in mind and a scan of the smear quickly suspected Streptococcus anginosus group as the culprit in this infection.

Some background;

In 1956, microbiologist W. D. Miller began to isolate and group a number of oral streptococci which later became known as the Streptococcus milleri group, named in his honour. Being streptococci they are gram positive, catalase negative cocci occurring in chains however the milleri group exhibited considerable variability in hemolysis, Lancefield group antigen and other properties typically employed in streptococcal identification. Recently, with the advent of DNA analysis, it was determined that this group consists of three distinct species designated S. anginosus, S. constellatus and S. intermedius. This rearrangement of the S.milleri group is now known collectively as the Streptococcus anginosus group. The historical name of Strepticoccus milleri group, however, continues to remain popular in Britain and parts of Europe.

The Streptococcus anginosus group of organisms are found as normal flora, particularly in the mouth and gastrointestinal tract. As opportunists they have been implicated in systemic infections at multiple body sites and are particularly associated with abscess formation. As such, if isolated from blood culture sterile fluids or abscesses, it is critically important that these organisms not be dismissed as contaminants.

Specifically, alpha or gamma hemolytic members of the S.anginosus group might erroneously be dismissed as the less pathogenic viridians streptococci. Beta-haemolytic members of the S.anginosus group organisms may also be initially confused with S.pyogenes , S.agalactiae, S.equisimilis and large colony group Group ‘G’ streptococci. It is therefore imperative that Streptococcus anginosus group be considered whenever such colonies are evident on cultures of abscesses and normally sterile sites. A complete identification is warranted and the isolates should never be reported solely by their Lancefield grouping. Also, although many of the commercial identification systems, whether manual or automated have the individual Streptococcus anginosus group species in their databases, studies suggest that the accuracy of specific speciation might be as low as 75%. Again, because of the serious pathogenicity of this group, one must be absolutely certain of the identification in reporting.

Streptococcus anginosus species isolated from Abdominal Fluid (Below)

Clues that an isolate might be one of the S.anginosus group;

Alpha, beta or gamma streptococci isolated from normally sterile site such as blood*, tissue & body fluids.

Isolated from abscess.

Slower growing colonies that may not appear until 36 – 48 hours of incubation.

Colonies that may exhibit enhanced growth in CO2 or an anaerobic environment.

Small colonies on sheep blood agar, typically less that 0.5 mm diameter.

Colonies on agar media give off a characteristic caramel or butterscotch odour.

Gram stain (see below)

*isolating S.anginosus from the blood should alert the clinician that there is a focus (abscess) elsewhere in the body serving as the source of the bacteremia.

To remember the species comprising the Streptococcus anginosus group, just think of the acronym of America’s Central Intelligence Agency, the CIA where now the letters can stand for Constellatus, Intermedius & Anginosus.

Jumble of Gram Positive Dots within White Blood Cells in Abdominal Fluid
Are these Streptococci?? Do they offer a clue to specific identification?
(Click On Photo To Enlarge for Better Viewing)

Streptococcus anginosus
Gram Stain of isolated colony taken from Blood Agar Plate of above specimen
This more familiar appearance of Streptococcal cells is what is usually seen even in direct smears of species other than the anginosus group.

08/01/11: Added this photo of Streptococcus anginosus in a liver asperate (above)
Gram positive cocci in pairs and chains-the textbook description of Streptococcus.

Studies have suggested that there may exist a correlation between S.anginosus group’s specific species and site of infection or clinical manifestation. There appears to be a marked association between S.intermedius and infections of the central nervous system. S.anginosus is the species most frequently associated with the gastrointestinal and genitourinary tracts and S. constellatus was most frequently identified from specimens from the respiratory system.

A quick reference chart of some of the characteristics
the Streptococcus anginosus group may exhibit.

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Schistosoma mansoni & haematobium

2010-11-21T20:02:00.013-05:00

Trematodes=flatworms=flukes

Phylum=Platyhelmithes
Family=Schistosomatidae

Primary human species are;

Schistosoma mansoni
Schistosoma haematobium
Schistosoma japonicum
(S.mekongi & S.intercalatum encountered less frequently)

Schistosomes infect about 200 million people worldwide, particularly in endemic regions such as Asia, Africa and South America. They have plagued man throughout history with evidence of infection (ELISA) in Egyptian mummies from the predynastic period (3100 B.C.) as well as Schistosome eggs detected in mummified kidneys (1250 B.C. to 1000 B.C.)

Schistosomes are often referred to as 'blood' trematodes as they differ from other trematodes because they infect humans by penetrating intact skin to gain entry to the circulatory system rather than infection through ingestion. In other words, Schistosomes infect humans through direct larval (cercariae) penetration rather than the ingestion of metacercariae.
Schistosomes are also unique among the flukes in that there are both a male and female organisms.
Schistosoma eggs also lack an operculum which characterizes other fluke eggs.

Their life cycle is as follows;

Eggs in feces or urine are passed into water
Larvae are liberated and penetrate the intermediate host snail where they further develop.
Cercariae emerge from the snail while in the water
Cercariae penetrate the skin of humans in contact with the water
Larval migration begins through the circulatory system where they may enter alveoli to produce hemoptysis. Organisms mature in the liver before entering specific veins specific to the infecting species. (S.haematobium in veins of the bladder, S.japonicum in veins of small intestine & S.mansoni in veins of the large intestine)
Eggs are passed to continue the cycle.

Pathogenicity of Schistosomes depends on the host's immune response to the various stages of infection, previous host exposure and the worm burden.
Symptoms include cercarial dermatitis, acute schistosomiasis (Katayama fever) and related tissue egg deposition. Acute schistosomiasis begins when the adult female begins laying eggs.

In the circualtory system it is believed the organisms either becomes covered with host soluble blood group antigens, lipoproteins, or develops antigens similar to the host's so that it excapes the host's immune response. For this reason, adult worms in the veins evoke little immune response.

Symptoms may vary in intensity but can include malaise, fever, abdominal tenderness or hepatic pain.

Infection with S.mansoni or S. japonicum may cause diarrhea. S. haematobium causes hematuria.

Morphology;
Schistosomiasis should be considered with any patient from endemic areas who has had exposure to untreated water and presents with symptoms previously mentioned. Diagnosis is confirmed with the identification of Schistosome eggs recovered the patient.

All eggs are embryonated when passed
All eggs are easily differentiated by their appearance

S.mansoni eggs are large (110-170 µm), oval and have a lateral, 'rose thorn' spine.
S.haematobium eggs are large, oval (110-170 µm) and have a terminal (end) spine.
S.japonicum eggs are smaller (55-90 µm), round and have a 'crooked finger' spine.

S.haematobium eggs can be recovered from centrifuged urine specimens
S.mansoni & S.japonicum from fecal specimens although on occasion both may be recovered from urine as well.

Treatment;
Praziquantel is the drug of choice in treating schistosomiasis. O & P examinations should be conducted periodically for up to a year post treatement to ensure erradication.

Schistosoma mansoni egg in concentrate (X400)
(Click on photos to enlarge for better viewing)

Schistosoma mansoni wallpaper (1024 X 768)
(note lateral 'rose-thorn' spine on egg)

I once encountered S.haematobium in the urine of a young Egyptian child however it was prior to my attempts at documenting interesting specimens in photographs. I have never personally seen a S.japonicum.

Update; I recently took some photos of S.haematobium from a preparation obtained from our Pathology department. Unfortunately I don't have much information on this patient's history nor the stain used. However, it does make for a pretty photo!

Schistosoma haematobium
(Note terminal spine)
(Click on photo to enlarge for better viewing)

Schistosoma haematobium (X400)
(Terminal spine at bottom barely visible)
(Click on photo to enlarge for better viewing)

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Bacterial Vaginosis

2010-11-14T19:24:00.019-05:00

A number of nasties can infect the female genital tract. The Previous post dealt with the parasite Trichomonas vaginalis. Other common infections are Chalamydia infections, yeast infections and bacterial vaginosis. While Neisseria gonorrhoea can be isolated from the vagina, a cervical swab is a superior specimen for detecting gonnococcus (G.C.) I'll do a post on Neisseria gonnorhoea infection at a later date.

Bacterial vaginosis is characterized by a thin malodorous vaginal discharge due to an alteration of bacterial flora caused by a shift in the acidity (pH) of the vagina. It's referred to as a 'vaginosis' rather than an 'vaginitis' because 'itis' implies an inflamation which would be accompanied by a proliferation of white blood cells (wbc's). Here, in a bacteria vaginosis, the bacteria flourish without a significant increase in wbc's.

A several markers provide clues in the diagnosis of bacterial vaginosis.

Potassium hydroxide - a drop added to sample of the discharge produces a fishy odour due to amines released.
pH measurement - loss of normal acidity (generally considered >4.5) may suggest a vaginosis diagnosis, however it is not very definitive therefore not usually done in the lab.
Observation and bacterial enumeration of genera present in a vaginal gram stain. Vaginosis is characterized by a decrease by the lactic acid producing lactobacilli (hence the name) and an increase in the specific organisms, Gardnerella vaginalis, Mobiluncus species, and anaerobic organisms such as Bacterioides & Prevotella species. The relative proportion and or presence of these organisms can suggest bacterial vaginosis.
Clue Cells - the presence of epithelial cells stippled with gram variable bacilli is suggestive of bacterial vaginosis. In Scanning the gram stain, the presence of these epithelial cells coated with gram variable bacilli appear purplish in comparison to the regular pinkish-red epithelial cells present. Why these bacteria tend to adhere is not fully understood.

Specific description of bacterial forms present;

Probably the most noticeable bacterial form present in bacterial vaginosis is referred to as 'gram variable' bacilli. These are the Gardnerella vaginalis bacteria whose individual cells can stain both purple and red with the gram stain. That is, some entire cells may be either gram positive (purple-blue) or gram negative (pinkish-red) or both. They are relatively small or short cells. The irregular gram staining properties historically have contributed to placing the organisms in different genera. Previously called both Corynebacterium vaginale (a gram positive genera), and Haemophius vaginalis, (a gram negative genera), before being given it's own genera of Gardnerella. It is a somewhat fastidious organism.
Mobiluncus species - various species of the genus may be present in bacterial vaginosis and are observed, if present, as gram negative curved bacilli.
Anaerobic bacteria such as Bacteroides and Prevotella also are disproportionate and can be seen as smaller straight gram negative bacilli

The bacteria mentioned above may be present in the normal vagina in small amounts. When whatever causes the conditions to change, these bacteria can overgrow the normal flora and therby cause vaginosis.

Lactobacillus species are regular gram positive bacilli (purple-blue rods) which can vary in shape depending on the species and environment. The lactic acid they produce within the healthy vagina keep it slightly acidic which restricts the growth of other species of bacteria.

A guide for enumerating the proportion of the various bacteria forms present in a vaginal smear has been created and is known as the Nugent Score.
By scoring the various bacterial forms and noting the presence of Clue Cells, one can standardize the criteria for determining if the patient does indeed have bacterial vaginosis.

While bacterial vaginosis is a genital, or perhaps a sexual disease, it is not considered a sexually transmitted disease. Sexual contact is not responsible for it's presence nor is it spread through sexual contact. It arises from changes within host allowing for the disproportionate proliferation of native bacterial species.

Now for some photos of what is described above;

(Click on any photo to enlarge for better viewing)

Normal vaginal gram stain (X1000)
Gram stain showing a couple of epithelial cells and numerous gram positive lactobacilli (purple rods). Note too that there are few if any white blood cells.

Ditto

Normal Vaginal Gram Stain (100X)
All epithelial cells appear similar with no 'Clue Cells' which would appear bluish due to all the
Gardnerella vaginalis organisms adhering to the cells.

Gram of bacterial vaginosis
Gram stain (1000X enlarged) showing epithelial cell coated with gram variable bacilli (Clue Cell).
Note gram variable bacilli (Gardnerella vaginalis) , gram negative curved bacilli (Mobiluncus sp.) & regular gram negative bacilli. Straight gram positive lactobacilli as seen in the previous photo are absent.

08/10/11: As an afterthought I added this photo which may better illustrate what is referred to as a 'Clue Cell' in vaginal swab gram stains. Here two epithelial cells are seen however the one on the left is 'coated' with gram variable bacilli (Gardnerella vaginalis) making the cell appear purplish. On lower power scanning, these are usually seen with some frequency in a patient experiencing bacterial vaginosis.

A couple enlarged (1000X) gram stains of gram variable bacilli.
Small, short cells are gram variable -gram negative (pink), gram positive (blue) or both at once.

Gram Stain (250X)
Shows numerous epithelial cells with one epithelial 'Clue Cell' near center right of photo,
Clue Cell is coated with gram variable bacilli giving it a purplish-blue appearance.
Again, note absence of any significant numbers of white blood cells (wbc's)

Gram Stain (1000X) of Mobiluncus species
(Click on photo to enlarge for better viewing)

Mobiluncus species contribute to Bacterial vaginosis. They appear as gram negative (pink) curved bacilli - (look like pink parentheses)
Present are also two large pink epithelial cells
* * *
Just for comparison: Vaginal Gram Stain of Yeast Infection (1000X)
Note: epithelial cells with yeast cells (purple) and yeast pseudohyphae (long purple thread-like structure).

Bacterial vaginosis can be treated with the antibiotics Metronidazole (Flagy®l) or Clindamycin amongst other antimicrobials.

My Related Papers;

Relative Susceptibilities of Gardnerella vaginalis (Haemophilus vaginalis), Neisseria gonorrhoeae and Bacteroides fragilis to Metronidazole and Its Two Major Metabolites.

Metronidazole In Treatment against Haemohilus vaginalis (Corynebacterium vaginale)

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Trichomonas vaginalis

2010-09-28T14:51:00.018-04:00

(Parasite - Flagellate)

Trichomonas vaginalis is a human parasite which has worldwide distribution and is most commonly isolated from the female genital tract. In the male, Trichomonas vaginalis has been isolated from the prostate so it may be necessary to treat both sexual partners in order to prevent re-infection.

Trichomonas infection in the female may be suspected if a thin frothy white discharge is observed. Diagnosis is best made using a wet preparation from a freshly collected swab. The organism is sensitive to both temperature and drying so it should be sent to the laboratory as quickly as possible without refrigeration. On receipt, a drop of sterile saline is placed on a glass microscope slide and with the swabs contents expelled, it is cover-slipped and examined under low magnification (100-250X).

Trichomonas is roughly pear shaped and is between 7 to 23 µm long by about 5 to 15 µm in size. It normally has 1 posterior and 4 anterior flagella which provides a rather rapid and jerky motility which draws one’s attention when examining fresh preparations. Even in specimens that have been somewhat delayed in transit, an undulating membrane running along a portion of the cell, may be seen beating, An axostyle is also evident running the length of the cell. Other structures may not be evident on an unstained preparation. Trichomonas is only found as a trophozoite as it has no cyst stage.

* * *

Two Trichomonas vaginalis cells (center) seen in a wet prep of a vaginal swab

Trichomonas is site specific and when found in the human genital tract it is diagnostic for Trichomonas vaginalis. Care must be taken not to contaminate the swab with fecal material as the non-pathogenic Trichomonas hominis can be found in stools. Trichomonas tenax may be found as a commensal in the oral cavity.

Gram Stain of Trichomonas in vaginal swab
(note size comparison to wbc's)

Although it is possible for an experienced technologist to see Trichomonas vaginalis in a gram stain, it is not the preparation of choice. A Hematoxylin stain as employed for fecal material examination would stain Trichomonas however the simple wet prep remains both cheaper and quicker. Other tests such as monoclonal antibody, enzyme immunoassay and latex agglutination have been developed. Serological tests have not proven to be effective .
Treatment with Metronidazole (Flagyl) is usually effective although resistant strains have been described.

Short video of motile Trichomonas vaginalis cells in wet prep. Flagellar movement and undulating membrane motion occasionally evident. Will replace with a better video when I get a more active specimen.
(Click on Lower Right Hand of Control Bar to view Full screen)

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Mycobacterium tuberculosis in Gram Stain

2010-09-27T22:28:00.013-04:00

Mycobacterium tuberculosis
(In Gram Stain Of Peritoneal Dialysis Fluid)

So, you find yourself the lone technologist manning the micro lab during the evening shift. A dialysis fluid arrives for routine analysis and a gram stain is performed on a cytospin(1) of the sample. Under the microscope no bacteria are initially evident during repeated scans - yet something catches the technologist’s eye. It may have been the odd string of purple-blue dots or perhaps thin strands of clearing where the surrounding material uniformly retains the safranin gram counterstain.

An astute colleague recently encountered that very scenario and immediately knew something was amiss. She immediately suspected the presence of either Nocardia, or due the site, more likely Mycobacterium species. A fluorescent acid-fast(2) Auromine-O stain was performed to confirm her suspicious of presence Mycobacterium species.

The patient was a 72 year old Oriental gentleman with chronic renal failure receiving peritoneal dialysis. The specimen was sent to the provincial health laboratories the following morning where further analysis by AMTD(3) confirmed the identity as Mycobacterium tuberculosis (TB).
The diagnosis came as a total surprise to the doctor in charge.

Below are some photographs I took of this rather interesting specimen. Additional cytospins were made with more material deposited on the slide resulting in more Mycobacteria per field. The initial gram stain was even more challenging than what is seen in the gram stains that follow.

Gram Stain of Peritoneal Dialysis Fluid ;
(All photos 1000X Magnification)

Can you spot the purple-blue dots in the lower right quadrant of the photograph above?
(click on photo to enlarge)

If one examines the cell wall of a Mycobacterium under an electron microscope, it resembles the gram positive cell wall structure. Mycobacteria, however, have a high content of mycolic acid associated with the cell wall which resists staining by the traditional gram stain method. For this reason the gram stain is not routinely used to visualize Mycobacteria. Alternative stains are employed such as the Ziehl-Neelsen, or Rhodomine-Auromine stains. The Mycobacteria are stained using these stains and the high mycolic acid content resists decolourization using a mild acid-alcohol solution.

'Ghost Cells' A clue to the presence of Mycobacteria in a gram-stained specimen
(Click on photo to enlarge for better viewing)

One clue found in a gram stain that may suggest the presence of Mycobacteria are the ghost like cells. These appear as a clear (unstained) line in the shape and size of a bacillus and are due to the mycolic acids resisting retention of the gram stain. As the bacterial cells are not stained they appear as a clear line or 'ghost cell' surrounded by material retaining the counterstain. Look carefully at the photo above (click on it to enlarge for better viewing) and look for the clear ghost cells in the left. Focusing the microscope up and down may bring areas retaining the gram stain into better focus thereby revealing the parts of the cell wall staining purple-blue as seen on the right. (arrows point identical areas to where the Mycobacteria are visible under varying focus) [contrast this appearance with the 'dots' that Streptococcus anginosus may exhibit in direct specimen gram stains]

Gram stain retained as purple-blue dots spaced between clear areas of high mycolic acid content that resists the stain. Easy to overlook by the untrained eye.

A cytospin of dialysis fluid containing a larger amount of material resulting in more bacterial cells per microscopic field. Short rows of dots are visible, however they are unlike the chain of cocci as a Streptococcus would appear.

Mycolic acids also assist Mycobacterium's ability to survive. They are similarly responsible in resisting the uptake of antibiotics used to eradicate the organism. The also resist being engulfed and killed by macrophages - a cellular defense mechanism of the body.

(1) Cytospin - a fluid specimen is added to a miniature funnel that is clamped to a microscope slide above a blotting spacer. The assembly is placed in a special centrifuge which then, under force deposits the liquid, under force onto the microscope slide. Solid material such as cells and bacteria are concentrated and somewhat flattened onto the glass slide and excess fluid is wicked away by the blotting material. This microscope slide is then stained by the desired method and examined under the microscope. This maximizes detection of any bacteria if present in the specimen.

(2) Acid-Fast/Auromine-O -Auromine-O, Auromine-Rhodomine, and Ziehl-Neelsen stains are known as 'Acid-Fast' stains. The two former stains are fluorescent stains which glow a bright yellow to apple-green under a particular fluorescent wavelength as seen under a fluorescent microscope. The later (Z-N) is examined under a traditional light microscope and Mycobacteria will appear bright red against a green (malachite green) or blue (methylene blue) counterstained background. Mycobacteria resist being decolourized by a mild acid-alcohol solution and as they retain these stains, the cells are said to be 'acid-fast'.

(3) AMTD - An acronym for Amplified Mycobacterium tuberculosis Direct test. This is a DNA probe test that looks for a particular and unique sequence of nucleic acids within the microbes genome that only occurs in Mycobacterium tuberculosis. Finding its presence confirms the organism is Mycobacterium tuberculosis and not a 'MOT' (Mycobacterium Other than Tuberculosis). Other species of Mycobacteria exist and not all have the same devastating consequences assiciated with TB. AMTD is a very rapid test for identifying TB in a sample compared to the weeks to months required to isolate and identify the bacilli by conventional culture techniques.

Our fluorescent microscope does not permit the attachment of the camera at this time so I'm currently unable to take photographs of the bacilli fluorescing in the acid-fast stain employed by our laboratory.

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* * *

Cryptococcus neoformans

2010-09-06T18:08:00.011-04:00

Cryptococcus neoformans
Yeast

Worldwide distribution often found in soil contaminated with bird excrement, in particular, pigeon droppings have frequently been implicated.

Cryptococcus neoformans is a rapidly growing typically round yeast (5-10µm) capable of producing polysaccharide capsules which often surround the cell. A 1% peptone solution might aid encourage capsule production. Some species &/or strains of Cryptococcus will not produce capsules in-vitro. Capsule production is best demonstrated using the India-ink (or Nigrosen) negative staining technique. Colonies producing capsules on culture are often evident by their glistening wet or mucoid appearance whereas colonies which fail to produce capsules or have diminished ability to form capsules typically produce dull, creamy, butyrous colonies. Cryptococcus neoformans can be sub-typed further based on serological response to capsular antigens.

Click on Photo to Enlarge

Characteristics;
Urease Test Positive: (urea split to release ammonia which raises the pH of the phenol red indicator in the media turning it from a straw colour to a bright pink.)
C.neoformans and other Cryptococci inhibited by Cycloheximide.
C.neoformans can be differentiated from other Cryptococcus species using the Caffeic acid test (a substance used as a substrate to demonstrate phenoloxidase activity. If phenoloxidase is present, it breaks down caffeic acid to melanin with resulting brown-black colour production.) Note: phenoloxidase is inhibited by the presence of glucose so culture the yeast on a glucose-free media such as Cornmeal-Tween 80 agar.
C.neoformans grows well at 25oC as well as 37oC. Some other Cryptococcus species will not grow at 37oC.
Cryptococcus neoformans & most other Cryptococcus species do not produce pseudohyphae.

Clinical Manifistations;
Cryptococcus infections can be found with increasing frequency amongst HIV patients and others who are immunocomprimised however Johns Hopkins Medical center has noted an interesting pattern regarding the serotypes of C.neoformans.
C.neoformans v. neoformans is the most common, usually afflicting immunocompromised hosts while C.neoformans v. gattii is most common in immunocompetent hosts.

Cryptococcus neoformans infection is primarily acquired through inhalation and may invade;

Respiratory system - (Sputum, Broncheal Wash, Lung Biopsy)

Central Nervous System (CNS) - (Lumbar Puncture)

Also implicated in;

Skin infections

Bone infections

Other sites (disseminated)

Treatment; (dependent on site of infection)
Amphotericin B
Flucytosine
Fluconazole

Prognosis varies.

The photos on this post were taken from speimens obtained from an 80 year old woman of Indian heritage who presented with respiratory distress. Unfortunately she succumbed to her infection.

Gram stain of sputum specimen 1000X showing cell & size variations
(Inset: Top -Cell showing Capsule, Bottom -Budding cell and clear capsule)

India Ink preparation taken from SAB isolate showing numerous Cryptococcal cells surrounded by clear capsule (negative staining)
(Inset: Enlarged photo of Cryptococcal cell & budding daughter cell surrounded by clear capsule)

Rather unremarkable round Cryptococcal cells taken from Cornmeal Agar Plate

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Chaetomium Species

2010-08-14T10:56:00.020-04:00

Chaetomium Species
Fungus

Had some fun playing with this fungus in getting the ascoma (perithicium) to develop for these photographs. Still trying a few tricks so additional photos may be posted later.

In the clinical laboratory, media available is geared towards economical, rapid and efficient identification of the most common human pathogens. Unfortunately, resources (specialized media) are scarce if one wishes to experiment with ‘environmental’ organisms. Although generally considered to a saprophyte, these days virtually any organism can be responsible for disease in the imunocompromised host. Chaetomium has been implicated as agents of onychomycosis, peritonitis as well as having caused cutaneous lesions.

There are over 180 known species of Chaetomium, many of which are active in the breakdown of cellulose in the environment. Items such as paper or textiles in contact with soil, straw, dung etc decompose in part, due to the action of Chaetomium.

I believe the species I have here is Chaetomium globosum, one of the most common and widely distributed species of Chaetomium.

Macroscopic;
Pale yellow to a greyish-green depending on media and length of growth. Relatively rapid growth. Does not grow at 42 Celcius.

Microscopic;
Hyaline septate hyphae. Ascoma (Perithecia) are spherical to ovoidal to obovoidal (175 - 200 µm in length) with numerous hairs, usually unbranched, flexsulose, undulating or coiled, septate, brownish in colour and up to 500 µm in length.
Asci are clavate (30 - 40 X 11 - 16 µm in size) containing eight brownish limoniform (in face view) ascospores 9 - 10 X 8 - 10 µm in size.

Photographs below were taken using sticky-tape preparations, slide cultures on SAB media as well as growing the fungus on somewhat nutritionally deficient Corn Meal Agar, partially covered with a cover slip to vary the atmospheric tension. Ascoma were best seen on the slide culture and at the edges of the cover slip after about 14 days of incubation.

Still haven’t managed to induce and photograph the asci and ascospores. From what I understand, the fruiting of Chaetomium in culture can be stimulated by the addition of cellulose in the form of filter paper, cloth or jute fiber. It seems that compounds excreted by the fungus Aspergillus fumigatus can stimulate fruiting as well. Sugar phosphates and phospho-glyceric acid, by-products produced by A. fumigatus have been shown to stimulate the production of asci and ascospores. Calcium may also have an effect on fruiting. Perhaps I’ll play around with a few of these to see if I can induce fruiting structures for future photographs.

Plate at right is Chaetomium on SAB after about 96 hours incubation at 30 C.

Chaetomium in slide culture growing on edge of corn meal agar as seen after about 14 days incubation at 30C (X100)

Chaetomium ascoma on Corn Meal Agar at about 10 -14 Days (x100)

Chaetomium ascoma

I just think they look cute! Only a microbiologist would understand...

Lacto Phenol Cotton Blue (LPCB) Tape Preparation

Chaetomium ascoma and ascospores ejected at top

Slide Culture (SAB) ~12 Days -x100

Intended as Wallpaper (1024 X 768) -May be re-sized by Blogger

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Klebsiella pneumoniae Capsule

2010-08-10T22:24:00.005-04:00

Klebsiella pneumoniae Capsules
Bacteria (Gram Negative Bacillus)

In a previous post I discussed the Quellung Reaction which is used to visualize capsules produced by the gram positive organism Streptococcus pneumoniae (pneumococcus), There I mentioned that organisms other than the pneumococcus are capable of producing a capsule that surrounds the bacterial cell. Bacteria strains of E.coli, Klebsiella pneumoniae, Haemophilus influenzae, Neisseria meningitidis, Pseudomonas aeruginosa and Cryptococcus neoformans are capable of capsular production.

Bacterial capsules, often referred to as the Glycocalyx, are generally composed of polysaccharides with a high percentage of water, however other materials such as polypeptides and hyaluronic acid may be components.

Capsules may be beneficial to the cell because;

Capsules may provide a virulence factor by protecting the bacterial cell from destruction by phagocytes, thereby allowing it to survive and spread.

The high water content may protect the cell from dehydration, from hydrophobic agents such as detergents and their own parasitization by bacteriophages (viruses). The high water content also makes the capsule difficult to stain with conventional microbiological stains.

Although the capsular polysaccharides are difficult to stain directly using stains common to the microbiological lab, they may be visualized indirectly through negative staining(1). This may be done deliberately(2) or may be seen as an incidental effect through a routine staining process.

Below is a routine gram stain of a patient’s sputum specimen. On examination the presence of numerous coliform-like gram negative bacilli were noted. Occasionally, where conditions were just right, the dark red (gram negative) colour of the bacterial cell and the varying shades of the sputum can be seen separated by a small zone. This is the bacterial capsule and its presence in this case, along with the size of the bacterial cell, would provide immediate suspicion that this organism may be Klebsiella pneumoniae. Subsequent culture confirmed the initial suspicion of the organism responsible for this respiratory infection,

Here the capsule is visualized without using antibodies as with the Quellung reaction with Streptococcus pneumoniae. Antibodies against K. pneumoniae could be produced to enhance appearance of the capsule. These may be manufactured and utilized in research however they offer little to the diagnosis and management of K. pneumoniae in the clinical setting, The cost would preclude their use in routine diagnosis.

Streptococcus pneumoniae’s capsule may also be seen in the gram stain where conditions are right however, quick and confident diagnosis justifies obtaining and using Quellung antisera.

Gram Stain of Sputum Specimen Showing Capsules Surrounding a Gram Negative Bacillus

(1) Negative staining is a technique where you don’t stain what you wish to visualize but rather you stain everything around it thereby enhancing the contrast and making the object more visible.

(2) An example of deliberate negative staining - using Nigrosin (or India Ink) to visualized the capsule surrounding the yeast Cryptococcus neoformans. In a darkly stained field, the yeast cells would be seen as a natural or neutral colour, with a definite clear zone seen around the outside of the cellular wall.

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Strongyloides stercoralis

2010-07-29T23:28:00.011-04:00

Helminth; Nematode (Parasite)

Disease; Strongyloidiasis

Geographic Distribution;
Worldwide distribution but more prevalent in warm climates and where the groundwater is high. Endemic in the tropics and sub-tropics as well as the South-eastern United States, particularly Kentucky and Tennessee. The geographic range is found to overlap that of the hookworm.

Life Cycle;
Strongyloides has a somewhat more complex life cycle. Infection is usually acquired by skin penetration of the infective filariform larvae. The larvae are long and slender up to 630 µm in length by 16 µm in width. After penetration the larvae are carried by the circulatory system to the lungs where they migrate into the alveoli. (other routes may exist) Migrating up the respiratory tree they reach the trachea and pharynx where they are swallowed to then travel to the duodenum and upper jejunum. After about two weeks of development, the female begins producing eggs. Thin shelled oval eggs are roughly 50 to 59 µm in length by 30 to 34 µm in width, making them a bit smaller than hookworm eggs. Eggs hatch to release non-infective rhabditiform larvae that pass out of the intestinal tract via feces. Once in the soil they develop into free living male and female worms where they eventually produce infective filariform larvae.

Strongyloides stercoralis larvae (fecal concentrat X400)
(click on photo to enlarge for better viewing)

Autoinfection can occur with Strongyloides. Rhabditiform larvae within the intestine can develop into the infective filariform larvae as they pass through the bowel. These larvae can then reinfect the host by once again penetrating the bowel, traveling through the circulatory system to the lungs where they once again cycle through to reach the intestine or be finally passed out of the body in faeces.

Strongyloides stercoralis larvae in fecal concentrate (X250)
(click on photo to enlarge for better viewing)

Strongyloides stercoralis larvae (Iron Hematoxylin Stain X250)
(click on photo to enlarge for better viewing)

Rhabditiform larvae that pass in the stool usually measure up to 380 by about 20 µm wide. There is usually a readily visible genital primordial that occurs about two-thirds of the way down from the anterior end. Strongyloides and Hookworm larvae appear similar however on closer examination it becomes obvious that the buccal cavity of S.stercoralis is quite short while the mouth of the Hookworm rhabditiform larvae is about three times as long.

Strongyloides stercoralis larvae (anterior end) showing short buccal cavity (X1000)
(click on photo to enlarge for better viewing)

Strongyloides stercoralis showing genital primordium (Iron Hematoxylin X 1000)
(click on photo to enlarge for better viewing)

In the human the infective cycle can have three areas of involvement.

Skin (cutaneous) - usually little reaction by the host except for possible pruritis & erythemea with larger infecting numbers.

Pulmonary (lungs) - may be asymptomatic or present with pneumonia-like symptoms. Symptoms may include coughing, wheezing, shortness of breath, fever and pulmonary infiltrates. (Loeffler’s Syndrome). Sputum samples can be examined directly for the presence of larvae.

Bowel (intestinal mucosa) - symptoms may mimic a peptic ulcer with accompanying abdominal pain. May also mimic Crohn’s disease.

In chronic disease cases, the host and parasite reach an equilibrium - both co-exist with little or no detriment. Undiagnosed disease can persist in travelers some 30 to 40 years after having returned from endemic areas. Infection may not be evident until some event upsets the equilibrium, allowing the parasite to flourish. Immunosuppresive therapy may predispose an individual to this hyper-infective syndrome.

In addition to tissue damage caused by the migrating larvae, Strongyloides infection may result in bacterial sepsis caused by the extra-intestinal dissemination of gut flora. Peritonitis, brain damage and respiratory failure may also be consequences of infection.

Identification;
Confirmation of infection is accomplished by recovery of adult worms, larvae or eggs in the stool or sputum. Stools may vary day to day with the presence of Strongyloides often making it a challenge to diagnose even in concentrates. Duodenal aspirates and Entero-Test capsules provide alternative sampling techniques which may or may not be effective.
If only filariform larvae are recovered, Strongyloides larvae may be confused with Hookworm larvae. One distinguishing difference between the two larvae is that the tail of Strongyloides is notched while the hookworm is sharply pointed.

Strongyloides stercoralis egg containing larvae (fecal concentrate X400)
(click on photo to enlarge for better viewing)

As above but Iron Hematoxylin Stain (X400)
(click on photo to enlarge for better viewing)

In heavy infections eggs may be recovered from the stool as well as both rhabditiform larvae and filariform larvae. Adult worms may also be present.

Female Strongyloides stercoralis (Iron Hematoxylin Stain X250)
(click on photo to enlarge for better viewing)

Therapy;
Repeated doses of thiabendazole may be required to cure a Strongyloides infection. Cure rates range from 55 to 100 percent. Patients should be screened for Strongyloides infection prior to receiving immunosuppressive drugs.

Epidemiology and Prevention;
Hygene is all important in preventing Strongyloides infection. Avoiding contact with contaminated soil, surface water and feces will break the infective cycle.

Link to my old post on Strongyloides stercoralis with single photo taken with film camera.

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Aspergillus nidulans (Emericella nidulans)

2010-07-18T10:20:00.013-04:00

Aspergillus nidulans
Filamentous Fungus

Ecology - Widespread, ubiquitous soil organism found most frequently in tropical and sub-tropical climates.

Macroscopic:
- Colonies rapidly growing, green to cream-buff.
- Dark green to orange and yellow in areas with Cleistiothecal production.

- Brown to purplish exudates. Purplish reverse.
- Moderate to rapidly growing.
- Colonies dense, velutinous with floccose surface.

Microscopic:
- Conidial heads radiate to loosely columnar.
- Conidiphore stipes 60-130 X 2.5 -3.0 μm.
- Phialides biseriate, limited to upper surface of the vesicle.
- conidia round, smooth to rugulose, (3-4 μm dia.) in short chains
- Cleistothecia often numerous, reddish-brown in colour.
- Hülle cells (10 - 25 μm dia.) typically present. Thick walled, smooth, often surrounding Cleistothecia.
- lenticular ascospores (within Clesitothecium), typically reddish-purple with two equatorial crests. Ascospores (4-6 X 3-4 μm) mature within 2 weeks.

Misc:
-Teleiomorph = Emericella nidulans (Cleistiothecium = sexual state)
- Mycotoxin produced = Sterigmatocystin

Conidiphores Bearing Conidiospore chains, Also Hülle Cells
(Tape Preparation, LPCB X250)

Smooth Round Hülle Cells (10 - 25 μm dia.)
(X400, LPCB)

Emericella nidulans - Split Cleistothecium Releasing Ascospores
(Tape preparation in KOH - natural purple colour due to reaction of Cleistothecium contents with KOH -X400)*

Ascospore Contents of Cleistothecium With Hülle cells Present
(natural brownish colour of Ascospores and purple colour of those reacting with KOH)

(Intended as 1024 X 768 Wallpaper - May be re-sized by Blogger)
Aspergillus nidulans/Emericella nidulans
(Cleistothecium surrounded by Hülle cells, expelling ascospores)

*Make an adhesive tape preparation of Emericella nidulans (Cleistothecia) substituting KOH for LPCB. Once clestothecia located under the microscope, swing objective out of the way and use the eraser end of a pencil to press down on the tape thereby breaking cleistothecia open. Purple colour develops immediately when contents (ascospores) come in contact with the KOH base. May take several attempts to 'squash' , locate and refocus.

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Aspergillus species Foot Cell

2010-06-30T15:59:00.005-04:00

Aspergillus (species in general)
Fungus

I came across this excellent example of an Aspergillus species foot cell and wished to share it. I couldn't restate the description any better that what already appears in the text below. (see footnotes for source)

"During mycelial differentiation certain cells enlarge, develop a heavy cell wall and form ‘T’ or ‘L’ shaped ‘foot cells’ (which are not separate cells) that produce a single conidiophore perpendicular to the long axis of the cell. Sometimes it is difficult to see the foot ‘cell,’ but when visible, morphologists take it as strong evidence that an isolate is an Aspergillus species. The erect hyphal branch developing from the foot cell is the conidiophore, which enlarges at its apex to form a rounded, elliptical or club shaped vesicle. (1) "

Aspergillus species foot cell seen at base of conidiophore (arrow)
(click on photo to enlarge for better viewing)

Aspergillus species foot cell seen at base of conidiophore (arrow)
(click on photo to enlarge for better viewing)

(1) Text Source Quoted: Aspergillus: Molecular Biology and Genomics
Edited by: Masayuki Machida and Katsuya Gomi
Published: 2010

(Photos are mine)

For further reading see also;
Mycopathologia
Volume 49, Numbers 2-3, 209-215,
Ratna Singh

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Quellung Reaction (Streptococcus pneumoniae)

2010-06-29T20:58:00.006-04:00

Bacteria
Streptococcus pneumoniae = pneumococcus

Quellung Reaction

Around the turn of the century (1900) German physician and bacteriologist Friedrich Neufeld discovered that antibodies against specific pneumococcal capsular antigens could be produced and used for typing Streptococcus pneumoniae organisms. These antibodies, when bound to the cell wall antigen produced a clearing around each individual cell with the appearance of the capsule having swollen. The word “Quellung” is German for ‘swelling’ however this is a misnomer as the capsule does not swell but simply appears enlarged with the clear zone produced by the bound antibodies. The clearing is best visualized by using a stain to enhance contrast between the clear zone of bound antibody and the surrounding material. Methelyne Blue is commonly used as in the photo below.

A patient’s specimen, often sputa, cerebral spinal fluid (CSF) or colony isolate, is placed on a clean microscope slide and allowed to dry. A drop of Polyvalent(1) Quellung antisera is applied to the specimen/slide and mixed with a drop of methyene blue stain. A coverslip is place onto the mixture and allowed to incubate/react at room temperature for 15-20 minutes. (can be placed in a humidity chamber to prevent evaporation). The slide is then examined under the microscope for distinct clearing around the cells. One may have to search for a microscopic field which best shows the clearing. A distinct zone of clearing indicates a positive Quellung reaction and confirms the identification as Streptococcus pneumoniae.

The price of Quellung antisera has escalated in recent years (~$600/ml CND). A limited shelf life and prohibitive cost make its use rather restrictive. Molecular techniques may soon replace this classic test.

Specific antibodies can be produced against the capsular antigens of organisms from other genera that have capsules -eg. Klebsiella pneumoniae.

1) Polyvalent Antisera- contains all known serotypes to identify any pneumococcus. Specific or individual antisera can be employed for scientific study of individual serotypes.

Click here for an actual Case Study located in this Blog

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Rhizopus species

2010-06-29T20:37:00.009-04:00

Fungus

Rhizopus is a cosmopolitan filamentous fungus found in soil, as a saprobe growing on mature fruits & vegetables and as mold found on old bread.

The genus Rhizopus consists of a number of species exhibiting many common features. An adhesive tape preparation(1) stained with Lactophenol Cotton Blue for contrast is shown below.

Asexual sporaniospores are borne from a sporangium located upon the collumella at the apical end of the sporangiophore. Rhizoids, for which the genus is named, appear as a root-like structure extending near the stolon hyphal base. Hyphae are aseptate or sparsely septate.

Macroscopically, Rhizopus is a rapidly growing fungus that can fill a petrie dish with fluffy, cotton-candy like growth in under 5 days. Growth is generally whitish in colour which can turn brown with age as a result of the maturation of the sporangiospores within the sporangium.

Rhizopus is grouped with other fungi of the Zygomycota phylum which cause similar infections commonly referred to as zygomycosis. This opportunistic infection can invade a wide variety of sites/tissues and occurs most commonly in diabetics suffering ketoacidosis and in immunocompromised individuals.

Treatment of zygomycosis may require surgical intervention; debridement of necrotic tissue where possible followed by treatment with Amphotericin B.

Click on photos to enlarge for better viewing.

Pigmented Rhizoid & Sporangiophore seen.
After spore release the apophyses and columella often collapse to form an umbrella-like structure as seen at the end of two of the sporangiophores above.

Mature sporangium releasing sporangiospores
(click on photos to enlarge for better viewing)

Ditto (rhizoid at base)

Sporangium with Sporangiospores at end of Sporangiophore
(click on photo to enlarge for better viewing)

Inset (top) Sporangium releasing sporaniospores
Inset (bottom) Sporangiospores (48hrs X400)

Intended as Rhizopus Wallpaper (1024 X 768) when posted.

1) Adhesive tape preparation - clear, sticky tape is pressed against a filamentous fungus colony, lifted and placed on a microscope slide on which a drop of Lactophenol Cotton Blue was placed. The phenol kills the fungus rendering it safe to examine outside of a laminar flow containment hood and the Cotton blue dye enhances contrast making features stand out for easier examination.

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Nocardia species

2010-06-29T00:14:00.014-04:00

(speciation pending)
Bacteria, with some fungal similarities

Bronchial wash specimens sent to our laboratory were examined microscopically by gram stain. Numerous gram positive branching bacilli were observed raising suspicion of Nocardia. A partial acid-fast stain (1) confirmed suspicions that the organism was indeed partially acid-fast and consistent with Nocardia. Gram stain results and presumptive diagnosis were reported to the doctor in charge in order to initiate immediate and appropriate antimicrobial therapy. The bronchial wash specimen was cultured on our routine media (Blood, Chocolate, & MacConkey agars) to which Sabaraud-Dextrose & New York City agars were added. Specimens were incubated in appropriate atmospheres/temperatures and because of the slower growing nature of Nocardia, the duration of incubation was extended past the customary 48 hours.

This bronchial wash specimen was obtained from an East-Indian gentleman who was experiencing coughing, chest discomfort, dyspnia (shortness of breath) and periodic bouts of haemoptysis (blood in sputum). The gentleman was otherwise healthy, worked as an accountant and had been a resident of Canada for quite some time. No predisposing factors were noted (ie. Immunodeficiency)

Interestingly, within a week we had a second case of somewhat rare Nocardiosis from an Oriental gentleman, the incident unrelated to the first.

In previous years, our lab would have speciated this organism ’in-house’, however with the current state of the economy and subsequent financial challenges, many of the necessary media and reagents necessary for further workup are no longer routinely stocked. Regretfully, in order to cut costs, infrequent or specialized tests are “farmed out” to our local Public Health Laboratory.

Nocardia has worldwide distribution with respiratory infections acquired through inhalation of contaminated dust whereas cutanious/wound infections acquired by traumatic injury. Immunocomprimised individuals are at greater risk of acquiring this opportunistic infection.

Nocardia is a strictly aerobic, catalase positive gram positive bacillus which can form filaments (reminiscent of fungal hyphae) and exhibit branching, The cell wall contains mycolic acids (found in Mycobacteria) which is responsible for a beaded appearance in the gram stain and partial acid-fast staining properties.

Nocardia infections have been shown to respond to treatment with trimethoprim-sulfamethoxazole (Septra), sulphonamides, ceftriaxone, and alizarin)

Below are representative photos of the gram stain, partial acid fast stain, and 5 day growth on NYC agar of the specimen described above. (Click on images to enlarge).

Broncheal Wash Direct Gram Stain
(Showing filamentous, branching gram positive bacilli)

Direct Partial Acid Fast Stain

Dry, Chalky Nocardia Colonies On NYC Agar at 5 Days

Nocardia Wallpaper (1024 X 768) *may be resized by Blogger

1) Partial Acid-Fast staining employed: Carbol fuscin stain (3 min), decolourize with 1% H2SO4 (until colour no longer comes off ~1 min) and counterstain with methyene blue (30 sec).

* * *
New 22/11/10
New 22/11/10 For Comparison (above)
Gram Stain- Nocardia species in Abdominal Abscess 87 yr old male
New 10/11/10-This Nocardia isolate, referred to our Provincial Health Laboratories has been identified as
Nocardia cryiacigeorgica

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Aspergillus niger

2010-06-13T19:10:00.010-04:00

(Fillamentous fungi)
Subgenus: Circumdati, Section: Nigri

This fungus was a bit of a challenge to photograph as it matured so quickly that conidia were often well dispersed when I was prepared to photograph the culture. It was difficult to find really good examples of the vesicles bearing metulae, phialides bearing intact conidial spores. Both adhesive tape preparations and slide cultures were studied.

Ecology; Ubiquitous, worldwide distribution, commonly found in mesophilic environments. One of the most common of the fungi in soil, rotting fruit & plant matter as well as many indoor environments. A.niger may be found as a common laboratory contaminant.

Macroscopic; Rapidly growing on Saboraud-Dextrose Agar starting with a white to yellowish felt-like mat of mycelia, quickly turning black as conida develop the pigment aspergillin during maturation. Reverse remains white to pale in colour.

Aspergillus niger on Sabouraud-Dextrose Agar 72 hrs at 30C
Black pigment from conidia maturing from center of colony
(click on photo to enlarge for better viewing)

Microscopic; Septate, hyaline (clear) hyphae. Conidiophores (Stipes) are long (400-3000 µm) with spherical vesicles at the apex measuring 30-75 µm. Aspergillus niger is biserate - metulae just about cover the entire surface from which the phialides extend. Conidia are globose, brown to black in colour, measure 3.5-4.5 µm in diameter and have a rough surface.

Large vesicle at end of broken conidiphore bearing metulae & phialides with black pigmented conidia already dispersed. Adhesive tape preparation was too disruptive in capturing structures intact. (LPCB X400)

Ditto (LPCB X400)
(Click on photos to enlarge for better viewing)

Aspergillus niger (LPCB X400)

Pathogenicity; not tremendously pathogenic however has been implicated in aspergillosis particularly in immunocompromised patients. Has also been isolated from ear infections (otomycosis), often as a secondary invader, establishing itself after/on top of a bacterial infection.

Industry; A.niger produces a number of useful enzymes which have been utilized by industry in the production of a variety of products such as citric acid. The possibility of A.niger being capable of producing mycotoxins remains controversial.

Treatment; Studies on the susceptibility of A.niger to antifungal agents remain inadequate. Isolates should be tested individually if therapy is warranted. Voriconizole as been shown to be effective in documented cases as well as Itraconazole and Amphoterecin B.

Intended as Aspergillus niger computer wallpaper (1024 X 768) when posted.
(click on photo to enlarge for better viewing)

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Enterobius vermicularis (Pinworm)

2010-06-13T17:11:00.013-04:00

Parasite (Intestinal Nematode)

Your kids wake up each morning with an itchy butt?? Perhaps they have a Pinworm (Enterobius vermicularis) infection. Pinworm might be considered to be the world’s most common human parasitic infection. Thought to be more common in cool and temperate regions where the climate doesn’t necessitate as frequent bathing and changing of clothing.

Infection is initiated by ingestion of eggs which migrate to and hatch within the cecum where they develop into adult worms. It takes about a month for the female worm to mature and begin egg production of her own. After the female has been fertilized, the male worms die off and may be passed in the stool. Gravid females may almost entirely be filled with eggs (See Photos).
The females migrate down the colon and out of the anus where they deposit their eggs on the perineal and perianal skin. Still under controversy is whether the female worms are able to migrate back to the intestine. This migration and resulting irritation (pruritus) causes an almost irresistible desire to scratch. While pinworm infections may be asymptomatic, itchiness is the most prevalent symptom reported. Eosinophilia may result, tissue invasion has been reported in a few cases and in females heavy infections may invade the genital tract. In children, the desire to scratch along with less vigilant hygiene, can result in re-infection and the infection of playmates through the oral-fecal route.

Worm Morphology;
The female worm measures 8 to 13 mm in length by 0.3 to 0.5 mm in width while the smaller male worm measures about 2 to 5 mm in length and 0.1 to 0.2 mm in width. Both male and female worms have bodies that quickly taper to a point (hence the name pinworm) however the female’s tale is straight while the male is curved at the caudal end.

Egg Morphology;
Microscopically, eggs appear to be shaped as miniature grains of rice, flattened on one side and measure 50 to 60 mm long by about 20 to 30 mm wide.

Diagnosis;
Paediatric patients presenting with non-specific complaints such as irritability or insomnia coupled with anal itching, should be examined for pinworm infection. Infection can best be diagnosed by using Scotch Tape™ or commercially available sticky paddles on which eggs and worms can be securely trapped. The optimal time to sample is in the early morning upon waking and prior to washing up for the day.
In the laboratory, the sticky tape is placed sticky side down onto a glass microscope slide and scanned at lower powers for eggs (or plate microscope -best for worms). Finding characteristic eggs and/or worms confirms diagnosis.
Female worms migrate on a sporadic basis so several (4 to 6) attempts may be necessary for conformation or dismissing as negative.
Treatment;
Mebendazole and Pyrantal Pamoate are effective in treating pinworm infections.
Epidemiology & Prevention;
Because infection is so common and easy to acquire, breaking the oral-anal route is the most effective way of preventing the infection. Better hygiene, frequent changing of bed sheets and night clothes as well as disinfection of toys, furniture and objects in close contact with those infected, reduce the risk of spread. The laboratory technologist is also at risk if the diagnostic sample is not handled appropriately.
Ultra-violet light, dry heat and chlorinated pools will kill Enterobius vermicularis.

Straight Tail of Gravid Female Enterobius vermicularis Worm
(Single egg outside of worm clinging to tail)

Higher Magnification of Worm Body Filled With Eggs

Yet an even Higher Magnification, outer wall of worm where writing appears above.
(Worm body is so packed with overlapping & stacked eggs that it almost appears opaque)

Three relatively clear, flattened rice shaped eggs appear in photo

Pinworm Egg in Fecal Concentrate X400

New; March 14, 2011 Haematoxylin & Eosin Sections of appendix from young child containing pinworms. The following photos wore taken using the Leica DMD 108 microscope,

Above; Four worms in transverse section seen in lumen of appendix. (X100)
(click on photo to enlarge for better viewing)

Lower left worm female showing ovaries containing developing pinworm eggs
Central worm has ruptured wall
All four worms have characteristic projections called 'alae' which on the worm furthest to the right I've highlighted with arrows.
All four worms have darker purple structure which is the intestine. The lumen of the intestine appears somewhat as an X-shape.

(click on photo to enlarge for better viewing)

Above; Female worm in transverse section showing two ovaries with developing eggs. Single projection (alae) appears in frame of photo on right side of worm. Intestinal cross-section seen in lower right of worm's interior. (X400)

(click on photo to enlarge for better viewing)

Above; Worm cut in somewhat a longitudinal section. Most probably appears to be anterior end of worm showing structure of which I am unsure. This might be the esophagus or the median bulb cut somewhat at an angle. (if anyone knows with certainty, post a comment). Two 'alae' are also seen at bottom left-of-center and top right-of-center of worm. (X400)

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Alternaria alternata

2010-06-13T14:29:00.010-04:00

Filamentous Fungus
(about 50 species of Alternaria are currently recognized.)

Alternaria is cosmopolitan and ubiquitous in nature. Primarily isolated from soil and from plants either as pathogens or as saprophytic contaminants.

Alternaria has emerged as an opportunistic pathogen primarily of immunocomprimised patients (transplant recipients, AIDs patients)
Alternaria has been recovered from several body sites (systemic & cutaneous) and can frequently be the cause of allergic fungal sinusitis.

Macroscopic;
- rapidly growing downy or cottony colonies maturing within 5 days.
- grey to olive brown on the surface with short aerial hyphae
- brown-black on reverse due to pigment production.
Microscopic;
- Alternaria produces the pigment melanin therefore structures can appear brown to black in colour.
- dark septate hyphae.
- large conidiophores (18-16 X 23-50 mm) are septate (transverse & longitudinal septations), simple or branched and occasionally exhibit a zigzag appearance.
- conidia (poroconidia) are brown, muriform, ovoid or obclavate, with an elongated ‘beak-like’ apical cell, solitary or acropetal chaining.

Notes;
Alternaria is distinguished from Ulocladium by
-obclavate conidia and a beak-like cell at the apex.
- conidiophores are comparatively less geniculate (bent) than those of Ulocladium.
-conidia are typically in chains (Ulocladium's found singly or in short chains.)

Photos that follow are of Alternaria stained with Lactophenol Cotton Blue (LPCB)

Alternaria mycelium as seen at 250X magnification

Note: Brown (melanin) Pigmented Septate Hyphae &
Both Longitudinal & Transverse Septations In Chaining Conidia

Intended as Alternaria Wallpaper (Sorry, Resized by Google Blogger)

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Whipworm (Trichuris trichiura)

2010-06-11T10:44:00.025-04:00

Helminth – Nematode - (Roundworm)

Trichuris trichiura (Whipworm) infections are most common in warm, moist areas of the world and are often seen in conjunction with Ascaris infections. Infections can be asymptomatic or symptomatic depending on the worm burden.
People exhibiting clinical symptoms may have abdominal pain, diarrhea, anemia and weight loss. Severe cases may result in rectal prolapses. Mechanical damage to the mucosal surface and allergic response by the patient appear to be the cause illness.

Worm Morphology;
Whipworms were named for their overall appearance – a narrow anterior with a thicker posterior giving the appearance of a handle with whip attached.
Whipworms measure from about 35 to 50 mm long with the male measuring somewhat shorter (30 to 45 mm), the male having a coiled tail.
The whipworm actually attaches itself into the lining of the cecum using hooklets at it's anterior (thin) end.

Life Cycle;
Unlike many roundworms, there is no circulation of the whipworm's larval stages throughout the body but is rather confined to the intestinal tract.
Human infection is acquired through the ingestion of fully embryonated eggs found in the environment. The eggs hatch in the small intestine and eventually attach to the mucosa of the large intestine. Adults mature in about 3 months and then begin egg production.
The eggs are passed in the feces in an infertile state and require about 10 to 14 days outside of the body, in a moist soil environment to embryonate and become infective.

Egg Morphology;
Trichuris trichiura eggs are 'football' or 'barrel' shaped with clear, mucoid-appearing polar plugs at each end. They are about 50 to 54 µm by about 22 to 23 µm wide.

Diagnosis;
Definitive laboratory diagnosis is made by demonstrating Trichuris trichiura eggs in the patient's stool. Eggs are best seen in stool concentrates but can also be seen in permanent stained smears. Stools preserved in PVA (polyvinyl alcohol) do not concentrate as well as those preserved in formalin and light infections may be missed. Adult worms are very rarely seen in stool samples.

Treatment;
Abendazole, although not as effective in treating whipworm as it is with other intestinal nematodes, it remains the drug of choice.

Epidemiology;
The geographic range of T. trichiura is similar to that of A. Lumbricoides and often the two infections are found together.
Infections are found most often in children as the eggs often are found contaminating the soil where they play.
Appropriate sanitation and disposal of feces helps break the reinfection cycle.

Whipworm segment in Histological Section

Another Section at Higher Magnification
(Note Trichuris Eggs Within Lumen of Worm)

Barrel or Football Shaped Egg (Fecal Concentrate)
(Note Polar Plugs -at each end)

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Giardia lamblia

2010-06-11T00:46:00.009-04:00

Parasite (Flagellate)

The flagellate Giardia lambila was first discovered by Leeuwenhoek in 1681 in his own stool but was not described in detail until 1859 by Lambl - hence the species named in his recognition.
Giardia lamblia has worldwide distribution and is the most commonly diagnosed intestinal parasite. It has an increased prevalence in children and daycare facilities probably due to the oral-fecal transmission.
Considerable debate continues on speciation and nomenclature of Giardia.
Giardia lamblia exists in two forms, a motile trophozoite form and the infective cyst form.

Trophozoites;
- multiply by longitudinal binary fission.
- most commonly found in the crypts of the duodenum.
- cyst formation takes place as the organism moves down through the colon.
- trophozoite is described as “leaf shaped”.
- trophs have four sets of flagella for motility.
- trophs have a sucking disc which facilitates attachment to mucosal surfaces.
- trophs have two nuclei, two axonemes, and two median bodies.
- trophs are approximately 10 to 20 m by 5 to 15 m in size.

Cysts;
- cysts are much more resistant to environmental conditions than the trophs.
- cysts are usually oval shaped but may appear round.
- cysts contain four nuclei, axonemes and median bodies.
- cysts normally measure 11 to 14 m by 7 to 10 m in size.

Clinical Disease;
- Giardia may be present in some without causing noticeable disease.
- Giardia lamblia is not penetrating/invasive but lives in the duodenal crypts and feeds off of mucosal secretions.
- in symptomatic cases there may be irritation of the mucosal lining causing diarrhea with resulting dehydration. There may be epigastria pain and increased flatulence.

Diagnosis;
- laboratory diagnosis is usually made by microscopically identifying the organism in stool samples. Both concentrates and stained smears are employed.
-cyclical shedding of the organisms made require a minimum of three samples taken over several days to ensure diagnosis.
- alternative methods such as a Entero-test capsule or ELISA tests are available.
- serological tests have not proven to be useful at present.

Transmission & Prevention;
- transmission is by ingestion of viable cysts
- contaminated food, water are frequently the source infection.
- found more frequently in people living in close quarters and in children that share contaminated fomites (toys, etc.)
- higher rates found in hikers, campers and canoeists that ingested contaminated water. Various animals such as the beaver can be the source of the organism, hence the slang term “Beaver Fever” often given to an outdoor acquired illness.

The illness may be self limiting however Metronidazole (Flagyl) is effective in eradicating the organism.

Microphotographs of Giardia lamblia (Below) stained with Hematoxylin & Eosin.

Enlarged Below

Cute little buggers, aren't they?

I always noted the similarity between the Giardia lamblia trophozoite morphology and Edvard Munch’s 1893 painting entitled ‘The Scream’. Perhaps Edvard had a bout of Giardiasis, the agony which inspired both the painting and title.

With apologies to Edvard Munch

New: December 2011
Another fine sample came my way and I thought I'd add a few pics of Giardia lambia trophs & cysts in an unstained concentrate as well as pictures of both trophs & cysts in the same photo stained with Iron Hematoxylin.

Giardia lamblia cysts in concentrate of stool specimen of patient with profuse diarrhea.
(click on photo to enlarge for better viewing)

Giardia lamblia trophozoite in concentrate of stool specimen of patient with profuse diarrhea.
(click on photo to enlarge for better viewing)

Giardia Cyst (Lower Left) and Giardia Troph (UpperRight)
Mixed bacteria & few yeast cells also present
Iron Hematoxylin (X1000)
(click on photo to enlarge for better viewing)

Giardia cyst (left) and troph (right) X1000
(click on photo to enlarge for better viewing)

3 Trophs & 3 Cysts
(click on photo to enlarge for better viewing)

Ditto; Two Pairs of Each
(click on photo to enlarge for better viewing)

Giardia lamblia in stool sample from patient with profuse diarrhea (X400)
(click on photo to enlarge for better viewing)

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Aspergillus terreus

2010-06-10T21:23:00.015-04:00

Fungus

This fillamentous fungus was isolated from a bronchial washing from on of our patients.

Aspergillus terreus is a cosmopolitan fungus although probably more prevalent in subtropical and tropical regions. In nature, it can be isolated from various sources such as grains, compost, straw, soil and decomposing plant material.

Macroscopic Appearance;
Colonies on Sabouraud-Dextrose Agar media appeared as rapidly growing powdery colonies with a characteristic buff or cinnamon-brown colour on the surface and a yellow to beige-brown colour on the reverse.

Microscopic Appearance;
-hyphae are septate and hyaline
-conidiophores are smooth walled and hyaline
-biseriate phialides, extending from the upper portion of the vesicle
-conidia form in long chains are round, smooth walled
-aleurioconida are less conspicuous and can be found on the submerged hyphae. Digging out a minute portion of the hyphae with a straight wire and squashing it under a cover slip may better reveal their presence. The appear singly as round to ovoid with truncate bases. Aleurioconidia production has been demonstrated to correlate with additional pathogenicity

Pathogenicity;
Species is a rare cause of allergic or invasive bronchopulmonary aspergillosis and noscomial infections have been reported. Implicated in a wide range of human infections from opthalmic, cutaneous pulmonary as well as disseminated infections. Increased morbidity and mortality in immunocompromised patients.

Demonstrated resistance to Amphoteracin B treatment.

Culture metabolites used in the pharmaceutical industry as a source for the production of statins employed in cholesterol lowering drugs.

Photos of 72 hour A.terreus stained with Lactophenol Cotton Blue under 400X magnification.

Intended as couple of Wallpapers however, resized by Google Blogger

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Helicobacter pylori

2010-02-06T14:55:00.005-05:00

Bacteria

Helicobacter pylori is a helical or spiral shaped bacterium which can infect the various areas of the stomach and duodenum. Infection can cause peptic ulcers, gastritis, duodenitis and perhaps even lead to cancer. Helicobacter pylori likes to live in the mucous layer of the stomach aiding it in surviving the stomach‘s acid environment. Helicobacter has enzymes which splits the urea molecule resulting in carbon dioxide and ammonia which further helps to neutralize and protect it from stomach acid.

Gastric biopsy tissue mashed onto glass microscope slide and stained with Basic Fuchsin Stain
X1000
(click on photo to enlarge for better viewing)

There are several tests developed to detect the presence of Helicobacter pylori. The laboratory can look for antibodies against the organism in the blood, Helicobacter antigens in the stool itself, detecting ammonia generated by the bacteria in a “breath test”, but the definitive test is by gastric biopsy via an endoscope down the throat.

Today therapy usually is by an antibiotic such as amoxicillin, clarithromycin or metronidazole and the inclusion of a “proton pump” inhibitor such as omeprzole.

The above photomicrographs are of a gastric biopsy. A very small (about 1mm square) piece of gastric tissue is mashed onto a glass slide, fixed with heat, then stained with Basic Fuchsin Viewed under a light microscope x1000.
(click on photo to enlarge for better viewing)

In my experience, Helicobacter pylori in gastric biopsy specimens can often appear very sporadically. One can search a slide for some ten minutes and just when ready to call the specimen "negative" you come across a field loaded with the characterisitic 'S' shaped cells. For this reason it is imperative to carefully examine the entire slide.

Click here to view my earlier post (2008) for H.pylori taken with film as opposed to digitally)

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Candida albicans

2009-12-09T18:40:00.002-05:00

Candida albicans
(Yeast)

I thought I’d start off my return from ‘sick leave’ by checking out the lab’s new digital camera. Just back, I have no “exotic” isolates to play with and explore so I’ve just retrieved a common, everyday Candida albicans. I’ve often, been disappointed in photographs found in most textbooks as they are small in size, black & white, poorly focused and often scrunched between a few lines of description. This yeast deserves a bit more respect as it is the most common cause of candidiasis, an infection which can range from acute, sub-acute or chronic. It may be present, yet unrecognized or have devastating, life threatening consequences. C.albicans may be present as commensal flora in the normal mouth, the skin and in the stool. Problems arise when, for what ever reason, the microbial balance is upset and Candida is allowed to overgrow other organisms which may assist in keep it in check. Antibiotic therapy may lessen normal bacterial flora allowing Candida to flourish. A change in the pH of the healthy vagina may result in a yeast infection. Immunosuppressive therapy may predispose a patient to infections by yeast.

Macroscopic;
Candida albicans grows rapidly in culture, reaching maturity in as little as three days. Colonies are cream coloured, raised, entire, smooth & butyrous. On enriched media such as Blood Agar, or Chocolate Agar, the colonies may develop small striations or outgrowths often referred to as “feet” which are indicative of the Candida albicans species.

Candida albicans on Sabouraud-Dextrose Agar at 48 hours at 30C
(click on photo and illustration at right to enlarge for better viewing)

Microscopic;
A smear made from colonies taken from Sabouraud-Dextrose Agar, or blood agar will appear as round to oval cells about 4 to 8 µm. Though the cell wall structure differs from that of gram positive bacteria, yeast cells retain the crystal-violet stain of the routine gram stain and therefore appear purple. The yeast cell divides by budding. On primary media (reduced nutritionally) the budding can create elongated cells which when lined up along the dividing plane, mimic the appearance of a hyphae however these inline individual cells are referred to as a pseudohyphae (false hyphae). Some true hyphae may also be formed.
Along side of the pseudohyphae, Candida albicans develops blastoconidia around the area of the ‘septa’ (division). These appear as smaller round ‘grape-like’ clusters.

Candida albicans retaining crystal violet stain from routine gram stain taken from SAB Agar
(click on photos to enlarge for better viewing) X1000

Another structure is the thick walled, rather refractle chlamydospore which usually develops at the end of the pseudohyphae (terminal).

Refractile Chlamydospore production on Corn Meal Agar at 2 hrs at 30C
(click on photo to enlarge for better viewing)

Technique; Chlamydospores production is best induced using the Corn Meal Agar (CMA) or Oxgall Agar. Inoculate the plate by picking up a sample of the Candida albicans colony with a straight wire and scratching the surface of the agar with it. With the surface inoculated, cover the scratched area with a microscope cover slip. This has a two-fold purpose. The glass cover slip reduces the atmospheric tension under the surface and protects the microscope objective from damage when viewing the plate after incubation. A filter paper moistened with sterile water and incubation at room temperature in the dark, may further encourage the production of chlamydospores After incubation (24-48 hours) remove the petrie dish plate cover (and microscope stage if you wish) and view the growth by carefully lowering the objective over the cover-slipped agar (X100-250). Viewing the growth around the edge of the glass cover slip should yield the best results.

Refractile Chlamydospores and smaller 'grape-like' Blastoconidia (Blastospores) produced on Oxgal Agar and photographed through the microscope cover slip with the agar plate placed on the microscope stage.
(click on photo to enlarge for better viewing)

Yet another characteristic of Candida albicans is that it has the ability to produce 'Germ Tubes' when placed in a nutritionally rich horse serum. Inoculate about 1 to 2 ml of horse serum and incubate at 37C for about 2 to 3 hours (too long may result in the formation of pseudohyphae, mimicking germ tubes) . Examine under the light microscope for a protrusion growing out from the yeast cell. Germ tubes appear as outgrowths from the side of the yeast cell and although characteristic of Candida albicans, be aware that the closely related Candida dublinensis can also produce germ tubes. Other characteristics which won't be discussed here can easily be used to separate these two species. Germ tubes protrude from the originating cell with no "pinching" seen at the point where they extend. If there is evidence of pinching, this may not be a germ tube but rather the beginning pseudohyphal growth.

Candida albicans germ tube production in Horse Serum at 37C after 3 hours incubation.
Note; there is no constriction/pinching where the germ tube leaves the originating cell indicating that this is indeed a germ tube and not the beginning of a pseudohyphae. (wet prep X400)
(click on photo to enlarge for better viewing)

Below are a few photos of Candida albicans as seen in actual specimens;

Candida albicans in a vaginal swab seen in the form of a pseudohyphae and a few individual oval cells. (Gram Stain X1000)
(click on photo to enlarge for better viewing)

Candida albicans seen as a pseudohyphae at lower left and individual yeast cells at upper right.
(Gram Stain of Sputum specimen X1000)
(click on photo to enlarge for better viewing)

Candida albicans pseudohyphae and individual cells seen in an aspirate from a kidney.
Depending of various factors, individual yeast cells, pseudohyphae, or both may be present in any given specimen.
(click on photo to enlarge for better viewing)

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Toys

2008-12-15T20:43:00.014-05:00

While I've always had an interest in photography, both macro and micro, inside and outside the laboratory setting, my talent has never measured up to my enthusiasm. Regardless I continue to try my best to document interesting specimens that I, or my colleagues encounter in the microbiology laboratory.

The photographs previous to this post were taken in entirety with a Leitz film camera head (right) mounted on the dual head Leitz microscope pictured below - all prior to the advent of digital imaging. Only the Tinea infected hair photo was taken using the same film camera mounted on a fluorescent microscope. Both Kodak Kodachrome & Ektachrome film of various speeds were previously employed.

In 2006 I suffered an injury which required prolonged rehabilitation and equally long absence from the microbiology laboratory. I started this blog out of boredom and posted older photos which I had on hand. On my return I found the lab had retired the old film camera and replaced it with a Nikon Coolpix 8400. The versatility of digital imaging was immediately realized particularly in allowing numerous shots with no waste of costly film & processing and instant feedback as to whether a particular feature of interest was adequately captured.

Above is the dual head Leitz microscope with Nikon Coolpix 8400 thatI've used for the majority of photos that follow in this blog. Prior to the lab's aquisition of the Nikon, this same microscope was outfitted with a Leitz film camera pictured top above right.

Microphotographs were fairly straight forward. The auto setting proved to be quite adequate for most stains and specimens. Flash was turned off. Photographs were captured in 300 dpi resolution and could be preserved or reduced in digital size or resolution as necessary. White balance could be corrected with photo editing software afterwords and cropped to focus attention on desired elements. Enhancements were kept to a minimum.

I quickly encountered several challenges when attempting macro photographs using the same Nikon 8400 in the laboratory setting, particularly in close-up photos of culture plates and media.

Photos taken at close range could not be taken with a hand-held camera as it could not be held sufficiently steady so as not to blur the photograph. A tripod was found to be too cumbersome as it was always a challenge to set it to the appropriate height on a bench top without having the legs splay out too far. Tripod legs set closer together often became included in the critical area of the photograph and were difficult to crop out. A tripod became all but impossible to set up within a biological safety cabinet when photographing fungal cultures.
Harsh overhead fluorescent lighting often produced both shadows and reflections from bench surfaces and clear plastic plate lids, frequently washing out the intended target.

As photography in our microbiology lab is almost exclusively of my own personal interest and the departmental budget does not currently permit purchase of a professional photo set-up, I improvised my own.
Modifying the spring-loaded articulated arm of an unneeded desk lamp, I constructed a mechanism that joined the lamp arm to the screw mount base of a spare tripod. The arm can be mounted using a clamp base attached to a workbench or when used in a biological cabinet, it is mounted in an alternate heavy movable metal lamp base. The spring-loaded arm can be swung into place without the base showing in the photo. The springs allow easy height adjustment, bearing the weight of the camera as it is raised or lowered in conjunction with the camera's zoom in order to obtain the ultimate combination. While the springs are too sensitive to motion to allow one to depress the trigger by hand, the Nikon 8400 comes with an infra-red remote controller. Once the camera is positioned, the spring-loaded suspending arm is released and vibration allowed to stabilize (just a couple of seconds) before snapping the photo. The movable base allows the arm to be moved a location in the lab with the best available lighting. The background can be selected to best contrast the item being photographed. All fungal plate photography is carried out in the biological cabinet so as not to contaminate the lab with fungal spores.

Above is my improvised spring-loaded lamp base with attached tripod screw mount allowing the camera position to be altered without getting the base into the photo. The heavy movable base allow the camera to be set up in a laminar flow hood for the safe photography of fungal cultures. A matt-black folder cover provides a disposable, non-reflective contrasting background for clear plates such as SAB. I rarely use the camera's flash as it tends to wash out images at the distances I require and reflections are hard to control.

New Toys (2010);
In the fall of 2010, our lab purchased a Leitz DMD-108 digital microscope. This headless (no occular) microscope utilizes LED illumination with the digital image being transmitted to a high-definition monitor. This unit allows for;

immediate monitoring of images by multiple observers displayed real time on the high-definition screen.
photography of any interesting field captured to a jump drive or other mountable storage medium.
record of camera/exposure settings for each image captured.
dictation of description of image features captured with built in microphone.
real time transmission of image over internet for consultation.
X & Y co-ordinates of image location for future orientation.
Digital zoom in addition to magnification provided by standard objectives.
White balance adjustment & colour correction for various common stains or custom settings.

While it has been exciting to obtain the Leitz DMD-108, I feel I have already encountered some limitations of this microscope. Immediately I noticed that it was extremely difficult to hold a field in focus when using the high oil immersion (1000X) objective. Perhaps the microscope still needs final set-up, however the scope was so sensitive that the image would drift out of focus immediately on release of the focus knob. This microscope was clearly better suited for lower magnifications where immersion oil was not employed. Oil immersion is most critical for the study of bacterial cells, limiting the usefulness of this instrument. Parasite species vary greatly in size and depending on which might require oil immersion or high-dry study. While permanent stained slides were easily viewed under high-dry power, wet preps of concentrates seemed to be difficult to follow as the suspension drifted across the field. Perhaps this requires some getting used to but suspended organisms seemed easier to follow on conventional microscopes. The same applied to fungal LPCB slide mounts however the larger size of conidiophorse and hyphae suspended in the more viscous LPCB reduced any focus and tracking challenges.
While an interesting instrument, it's usefulness in a microbiological setting is yet to be determined. The instrument seems better suited for histological/pathological specimens.

Above is a LPCB fungal specimen as seen on the high-definition monitor projected from the Leitz DMD-108 microscope seen on the right of the photo. Note the lack of occulars on this microscope.
In the future I will note any photos taken on this microscope. All others will continue to be taken using the Nikon 8400.

Tinea capitis:

2008-11-21T22:50:00.011-05:00

Fungus

Tinea capitis refers to dermatophytosis, a fungal infection of the scalp. Three types of in vivo hair invasion are recognized:

Ectothrix invasion is characterised by the development of arthroconidia on the outside of the hair shaft. The cuticle of the hair is destroyed and infected hairs usually fluoresce a bright greenish yellow colour under Wood's ultraviolet light. Common agents include M. canis, M. gypseum, T. equinum and T. verrucosum.

Single hair invaded by Tinea capitis (Calcofluor White Stain under Ultra Violet Microscope)

Magnification was not noted
(click on photo to enlarge for better viewing)

Endothrix hair invasion is characterized by the development of arthroconidia within the hair shaft only. The cuticle of the hair remains intact and infected hairs do not fluoresce under Wood's ultraviolet light which the physician might use in a presumptive diagnosis.. All endothrix producing agents are anthropophilic eg T. tonsurans and T. violaceum.

Favus usually caused by T. schoenleinii, produces favus-like crusts or scutula and corresponding hair loss.

Ditto

Tinea capitis infection of a single hair (Calcofluor White Stain under U.V. Scope)

The fungi that instigates tinea capitis thrives in moist and wet environments, and your risk increases if you have poor hygiene, prolonged skin wetness (such as that caused by sweating), and minor scalp injuries. Oral antifungal medication and medicated shampoos, such as those that contain selenium sulfide may be necessary to eliminate the infection.

The microphotographs above are of a single human hair with the arthroconidia visible within the hair shaft. They fluoresce because they were stained using Calcofluor White prior to being placed under a microscope cover slip and slide. Viewed under a fluorescent microscope at a magnification of x800.

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Neisseria menningitidis:

2008-11-20T23:57:00.002-05:00

Bacterium

Neisseria meningitides, also known as meningococcus is classified as a gram negative diplococcus, meaning it stains red using the gram stain and appears as two round cells in pairs. This organism is best known for its ability to cause meningitis. Symptoms may include fatigue, fever, headache, neck stiffness to coma and death can occur in about 10% of the cases. There are several serotypes (can produce distinct antibodies in response to their invasion) and these my correlate with the geographical location one aquires the infection. Antibiotic treatment might include Cephalosporins, Erythromycin, Tetracycline or Ciprofloxacin.

The photo above is of a specimen obtained from a Lumbar Puncture (LP) where a fine needle is introduced into the spinal column in order to obtain some Cerebral Spinal Fluid (CSF). The fluid was processed in a cytospin centrifuge which concentrates the specimen onto a small spot on a microscope slide, flattening out any white cells present in the process while drawing off excess fluid. White cells may be polymorph nucleocytes or monocytes depending on the infecting agent. The white blood cells are the body’s immune response attempting to defend it from the invaders. The slide is then “fixed” to adhere the material to the slide and then stained by the “Gram” method. Neisseria meningitides will appear as single or “diplo” (two) red coloured cells, often within the white blood cells. x1000 Magnification under a light microscope

Helicobacter pylori:

2008-11-20T00:01:00.008-05:00

Bacterium

Helicobacter pylori is a helical or spiral shaped bacterium which can infect the various areas of the stomach and duodenum. Infection can cause peptic ulcers, gastritis, duodenitis and perhaps even lead to cancer. Helicobacter pylori likes to live in the mucous layer of the stomach aiding it in surviving the stomach‘s acid environment. Helicobacter has enzymes which splits the urea molecule resulting in carbon dioxide and ammonia which further helps to neutralize and protect it from stomach acid.

There are several tests developed to detect the presence of Helicobacter pylori. The laboratory can look for antibodies against the organism in the blood, Helicobacter antigens in the stool itself, detecting ammonia generated by the bacteria in a “breath test”, but the definitive test is by gastric biopsy via an endoscope down the throat.

Today therapy usually is by an antibiotic such as amoxicillin, clarithromycin or metronidazole and the inclusion of a “proton pump” inhibitor such as omeprzole.

The above photomicrograph is of a gastric biopsy. A very small (<1mm dia) piece of gastric tissue is mashed onto a glass slide, fixed with heat, then stained with Basic Fuschin. Viewed under a light microscope x1000.

Histoplasma capsulatum:

2008-11-19T00:04:00.003-05:00

Dimorphic Fungus

Histoplasma is a thermally dimorphic fungus found in nature. Soil contaminated with bird droppings or excrements of bats is the common natural habitat for Histoplasma. Although it is claimed to exist worldwide, tropical areas are where this fungus is more frequently encountered. Histoplasma capsulatum is the causative agent of a systemic mycosis called histoplasmosis. The spectrum of the disease varies from an acute benign pulmonary infection to a chronic pulmonary or fatal disseminated disease. Inoculation is primarily through inhalation.

For definitive identification of the fungus, (>35C) yeast-to-mould (<35c). style="font-weight: bold; color: rgb(51, 204, 0);" size="4">

Diphylobothrium latum:

2008-11-18T00:10:00.003-05:00

Parasite

Diphylobothrium latum is also known as the broad fish tapeworm. There are many different host species, mainly those carnivores which eat fish, and this includes man. These worms can reach to a length of 10 meters and can shed in the region of one million eggs per day. The eggs are ovoid with an operculum (cap) at one end and a knob at the other and measure 60 x 40 microns in size. When they are released into the intestine they are only partially embryonated and require from 8 days to several weeks for the infective coracidium to develop. When the eaten by the definitive host it passes through the stomach and the scolex becomes embedded in the mucosa of the small intestine and develops rapidly producing eggs within 10-14 days. People generally become infected when uncooked fish are eaten and it is particularly prevalent in those cultures which eat a lot of freshwater fish and prepare it by methods other than cooking.

In many cases human infections go largely unnoticed, because of the nonspecific symptoms such as intestinal discomfort, nausea, and diarrhea. However, in some cases pernicious anaemia develops which is related to the malabsorbtion of vitamin B12.

There are a large number of possible drugs available to treat this disease, the two main ones used are Niclosamide and praziquantel, both of which are highly effective.

Effective control measures include cooking fish properly or freezing the fish down below -12 C for a minimum of 24 hrs.

The photomicrograph above shows the operculated eggs of Diphylobothrium latum with the pointer pointing at the cap. A wet preparation of a concentrated faecal sample was made. x800. Fecal material and bacteria can be seen in the background.

Anaerobiospirillum succiniproducens

2008-11-17T00:29:00.000-05:00

Bacterium

Anaerobiospirillum succiniproducens rarely isolated in the clinical laboratory. Septicemia appears to be the most frequently reported infection caused by Anaerobiospirillum followed by gastroenteritis. This strictly anaerobic, motile, gram-negative spiral bacterium is often mistaken for Campylobacter species when first encountered. Rapid identification is imperative as Erythromycin, effective against Campylobacter and antibiotics such as Penicillin or Metronidazole, frequently prescribed against anaerobic infections are ineffective against Anaerobiospirillum.

Entry into the body seems to be through the gastro-intestinal tract and may result in bacteraemia (bacteria in the blood). Gastrointestinal symptoms may include nausea, vomiting, abdominal pain, diarrhoea and rectal bleeding. Infection usually occurs when the patient already has some chronic underlying disorder.

The photomicrograph above is from a laboratory culture of the organism after it was initially isolated from the blood of a patient returning from overseas. A modified Leifson flagella stain was employed which stained the organisms red. The stain condensed around the very fine flagella making them visible under the light microscope. The flagella are whip like projections or tails which the organism uses to propel itself. Here you can see that there is a tuft of flagella on both ends of the bacterium. x1000 magnification.

Bipolaris specefara:

2008-11-16T00:36:00.000-05:00

Fungus

Bipolaris is a dematiaceous filamentous fungus. It is cosmopolitan in nature and is isolated from plant debris and soil. Bipolaris can infect both immunocompetent and immunocompromised host. Bipolaris is one of the causative agents of phaeohyphomycosis. The clinical spectrum is diverse, including allergic and chronic invasive sinusitis, keratitis, endophthalmitis, endocarditis, endarteritis, osteomyelitis, meningoencephalitis, peritonitis, otitis media (in agricultural field workers) and fungemia as well as cutaneous and pulmonary infections and allergic bronchopulmonary disease. Bipolaris can infect both immunocompetent and immunocompromised host.

The hyphae are septate and brown. Conidiophores (4.5-6 µm wide) are brown, simple or branched, bending at the points where each conidium arises from. The conidia, which are also called poroconidia, are 3- to 6-celled, fusoid to cylindrical in shape and are light to dark brown in color.

Amphotericin B and ketoconazole are used in treatment of Bipolaris infections. Surgical debridement may be indicated in some cases, such as sinusitis

The photomicrograph above has the indicator pointed at one of the typical poroconidia which is attached to it‘s conidophore from where it has branched off the hyphae. It is of a wet preparation stained with Lactophenol Cotton Blue and viewed under x800.

Entamoeba coli:

2008-11-15T00:39:00.001-05:00

Parasite

Enatamoeba coli is an amoeba which inhabits the human gut. It is considered to be an non-pathogenic commensal organism which along with thousands of other types of organisms makes up the normal gut flora. In the laboratory care must be taken in distinguishing the non-pathogenic Entamoeba coli from the pathogenic Entamoeba histolytica. Entamoeba can exhibit two forms, the trophozoite (troph) shown here and a cyst form which can exhibit up to 8 nuclei.

Treatment is not necessary but presence of this organism in a stool sample may indicate that a person has been exposed to a contaminated source bearing faecal matter and further investigation may be warranted.

The photomicrograph above is of an Entamoeba coli trophozoite in a concentrated faecal specimen stained by the Iron Haematoxylin method. A coarse cytoplasm is seen and the cell has a nucleus with dense unevenly distributed nuclear chromatin at it’s periphery and a central nucleolus. x1000.

Sporothrix schenckii:

2008-11-14T00:55:00.000-05:00

Dimorphic Fungus

Sporothrix schenckii is one of five species of thermal dimorphic fungi of medical interest. Dimorphic basically means “two forms” as the organism can express itself as a round cell yeast (>37C) or as a filamentous fungus (<37c).>
In nature Sporothrix lives as a saprophyte on wood, decaying vegetation, Sphagnum moss, animal excreta and soil. It can grow on the thorns of roses and can be inoculated into the body by a prick of the thorn. The fungus can then morph to a yeast and grow in the lymph system, manifesting itself as lesions in the lymph nodes. It is often referred to as “Rose Handler’s disease”. Sporothrix can also cause lung infections.

Antifungals such as Ketoconazole may have some effect. Itraconazole shows some promise at low doses for several months. Intravenous Amphotericin B, usually the drug of last resort for fungal infections, works poorly and slowly.

The photomicrograph above shows the fungal mycelia with typical hyphae bearing conidia. The “wet preparation” is dyed with Lactophenol Cotton Blue stain for contrast. x800.

Cryptosporidium parvum:

2008-11-13T00:58:00.000-05:00

Protozoan Parasite

Cryptosporidium is a coccidian protozoan parasite who’s oocysts can infect humans usually by the oral-fecal route. Contaminated water is often the source of infection. In recent reports improperly washed produce was implicated as the source of infection. In immunocompetent patients, cryptosporidiosis is an acute, self-limiting diarrhoeal illness lasting about 1 to 2 weeks. Symptoms may include frequent watery diarrhoea, nausea, vomiting, abdominal cramps with low grade fever. No safe and effective therapy for cryptosporidial enteritis has been successfully developed. Since cryptosporidiosis is a self-limiting illness in immunocompetent individuals, general, supportive care is the only treatment for the illness. In immunocompromised patients (AIDS, transplants, cancer patients, the illness may be much more serious. No effective treatment is available other than to prevent dehydration and let the illness run it’s course.

The above micro photograph is of a Cryptosporidium oocyst from a concentrated stool sample stained by Carbol Fuschin, then decolourized by acid-alcohol. The oocysts are “acid fast” and resist decolorization whereas other material loses the resulting red colour. The remaining material in the slide is fecal debris and normal intestinal bacterial. x1000.

Strongyloides stercoralis:

2008-11-12T01:02:00.000-05:00

Parasite: Nematode

Strongyloides stercoralis is a human parasitic roundworm (Nematode) about 2.5 mm in length. The adult parasitic stage lives in tunnels in the mucosa of the small intestine. The genus Strongyloides contains 53 species and S. stercoralis is the type species. The infectious larvae penetrate the skin when there is contact with the soil. Some of them enter the superficial veins and ride the blood vessels to the lungs, where they enter the alveoli. They are then coughed up and swallowed into the gut, where they parasitize the intestinal mucosa. S. stercoralis has a very low prevalence in societies where faecal contamination of soil is rare.

Ivermectin is the drug of first choice for treatment. Thiabendazole was used previously, but owing to its high prevalence of side effects and lower efficay, it has been superceeded by ivermectin and as second line, albendazole.

The above photomicrograph is of the Strongyloides larvae in a wet preparation of concentrated fecal material. x800.

Scedosporium inflatum:

2008-11-11T01:06:00.001-05:00

Fungus

Scedosporium inflatum is a filamentous fungus which is found in the severely immunodepressed including those with hematologic malignancy and recipients of allogeneic hematopoietic stem cell, heart-lung, lung, liver and renal transplants and occasionally in normal hosts as well. It can cause lesions in the sinuses, lungs, bones and central nervous system. The formation by S. inflatum of annelloconidia in wet clumps at the apices of annellides with swollen bases was found to be the most useful characteristic in differentiating this potential pathogen. Dissemination throughout the body might be prevented with combined itraconazole and fluconazole treatment. Variconazole is considered first-line treatment by some.

The above photo is of the hyphae and conidia stained with Lactophenol Cotton Blue and viewed x800.

E-coli & Ascorbic Acid Electron Micrographs

2008-11-10T01:09:00.006-05:00

Back in my university days I found employment at University Hospital in London at which I studied the effects of the antibiotic Metronidazole (Flagyl) against various bacteria (1). Metronidazole has long been established as one of the premier antibiotics with activity directed against most gram negative as well as many gram positive anaerobic bacteria. More interestingly, our research unexepectantly found that Metronidazole was able to exhibit remarkable activity against the facultative organism Gardnerella vaginalis (2). This was found to be a result of the metronidazole molecule being metabolized in the liver to both an ‘acid’ and a ‘hydroxy’ metabolite. Further experimentation showed that the hydroxy metabolite exhibited significant activity against this organism.

* * *

In the experiments using various anaerobic (oxygen hating) organisms, liquid tubed media was used to grow the bacteria and in which to dilute the antibiotic. In order to “reduce” the media, so that it was oxygen free, a variety of chemical ingredients could be added. We chose ascorbic acid and used the facultative anaerobic organism E-coli as a control. The test anaerobic organisms reacted as expected however significant changes were visible on the control E-coli bacteria as well, against which metronidazole should have no appreciable effect. The E-coli cells were visibly stressed and damaged, often showing uncharacteristic morphology such as incomplete cellular division, elongated cell forms and bifurcated ends. In a rather unexciting explanation, it was determined that this was due to the ascorbic acid content itself stressing the E-coli cells.

* * *

Regardless of this outcome, the entire process served as a learning experience and afforded me a chance to work with the university’s electron microscope. I’ve included some of those electron micrographs here. In particular I never tire of marveling at the physical and molecular structure of these fascinating bacteria. Included here is an electron micrograph of an E-coli bacterium demonstrating the cell wall structure which is responsible for it’s gram staining properties. Other smaller photos show the stressed nature of the bacteria which should have appeared as rather uniform bacilli. Various magnifications were taken and if I recall, the electrondense negative stain was phosphotunstic acid (PTA).

* * *

*all electron micrographs were taken by Yuri.

So, perhaps this is evidence that you should get your daily dose of ascorbic acid (vitamin C) as it may itself have a direct effect on bacteria in your system. You know what they say, “an apple a day keeps the doctor away” - perhaps with some truth to it.

(1) Edward D. Ralph & Yuri E. Amatnieks
Antimicrob Agents Chemother. 1980 March; 17(3): 379-382

(2) E.D. Ralph & Y.E. Amatnieks
Antimicrob Agents Chemother. 1980 July; 18(1): 101-104

Human Papilloma Virus (HPV) & Gardasil Vaccine

2008-10-31T18:35:00.005-04:00

I rarely agree with anything the monkeys in suits that constitute our government do, but this rare case is the exception. The Government of Ontario with the Ontario Ministry of Health have put together a program to educate and immunize appropriate citizens against infection with the Human Papilloma Virus or HPV.

Some Science First;

Papillomaviruses were first identified in the early 20th century, when it was shown that skin warts or papillomas, could be transmitted between individuals by a filterable infectious agent. In 1935 Francis Peyton Rous, who had previously demonstrated the existence of a cancer-causing Sarcoma virus in chickens, went on to show that a papillomavirus could cause skin cancer in infected rabbits. This was the first demonstration that a virus could cause cancer in mammals. Since then over a 100 different human papillomaviruses have been identified. Some cause benign wart or papillomas, after which the virus is named, but some 30-40 of these strains can be transmitted through sexual contact. Many of these infecting virus strains don’t cause any noticeable symptoms until greater problems arise, most notably, cervical cancer.

The papillomaviruses are non-enveloped, 60 nanometer (nm) diameter capsids composed of 72 capsomer units, geometrically arranged in icosahedral symmetry which then covers a double-stranded circular DNA molecule of about 8000 base pairs in length. The L1 protein of the Capsomers is what the HPV vaccine utilizes in eliciting antibody response against the invading virus, thus destroying it.

The Immunization Program & Stats;

The Government of Ontario has just introduced a $39M voluntary program in which all 84,000 grade 8 girls are eligible to receive the anti-HPV vaccine (Gardasil) free of charge in three doses over a 6 month period. The vaccine has been approved by Health Canada for use in girls and women between the ages of 9 to 26. The vaccine protects against infection from four separate strains of HPV, which combined, cause 70 per cent of all cases of cervical cancer.
Those not eligible for this program can still receive the vaccine at their doctor’s office at a cost of around $400.
Cervical cancer is the second most common cancer for women aged 20 to 44, after breast cancer.
In Ontario, some 550 women are diagnosed with cervical cancer and 150 die every year.

Side Effects;

As with any vaccination, the expected side effects, if any, include redness, tenderness and swelling at the injection site and less commonly, fever, nausea, dizziness and headache. No foreign substance introduced into the body can be considered 100% safe however serious side effects are extremely rare and outweigh the benefits.

The Controversy;

In this day and age it is still mind-boggling that vaccinations or anything related to sexual transmitted diseases can stir up such public controversy. You would think that any loving parent would do whatever they could to best ensure and protect their child’s health. However, prudish attitudes, outdated values and misinformed beliefs still supersede logic in many households.

Those opposed to their child receiving the protective vaccine usually present an argument such as:
Since this virus is only transmitted by sexual contact, my pure little grade 8 darling couldn’t possibly get this disease as she is not sexually active (and will never be until the night of her wedding). The twisted logic that also follows is that because their child would be protected from sexual HPV infection, she is more likely to engage in sexual activity now that the fear of disease (and God) are removed from the equation. These arguments are more likely to be raised by parents whose child attends a Catholic School or practice other more conservative religions.

It may be news to many parents of girls attending Catholic schools but the rate of promiscuity is about the same in their institutions as for the general public of the same age group. Peer pressure, the desire to rebel and “spread your own wings”, simple curiosity, society’s ever more liberal morals, and pure raging hormones, all may supersede any cautionary words spoken by a parent. Sadly, as remote as it seems, rape still remains a reality in this world. The result is that teens are engaging in sexual activity and ever increasingly younger ages. It is beyond me how any intelligent, loving parent in today’s society would deny their child this protection on misguided ethical or moral grounds. Education, instruction and proper parenting will not be diminished by your child having this vaccine. In the end, which would really be worse, a pregnant child or one that grows to develop a possible fatal cancer?

A final thought. Will your "pure as the driven snow" daughter who has remained celibate remember to get, and be willing to pay the $400 (current cost) for the vaccine prior to her wedding day? Just remember that your future son in law "Johnny Angel" may not have led such an chaste life prior to this blissful day and may be harbouring the virus. One final wedding day present for "your little girl". Think ahead, then act responsibly NOW!

No vaccine is 100% protective and being vaccinated does not eliminate the need to see your doctor for regular checkups including checking for cervical cancer.

* * *

Staphylococcus aureus

2007-09-06T20:03:00.000-04:00

Science and Staphylococcus aureus
(The little “BUG-ger” That Got Me)

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“Big bugs have little bugs on their backs to bite them, Little bugs have lesser bugs and so forth ad infinitum…”

Anonymous

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Microbiology. I first fell in love with the beauty and the science of microbiology while attending The University of Western Ontario (UWO) in London Ontario. My fascination with science began much earlier in a Hamilton public school. It was an epiphany when struck with the realization that the world around us does not behave randomly but rather is governed by strict rules and predictable properties which continue to be refined and discovered. From that moment on, my perception of the world changed. Science was all around us - everywhere, and all the time. My hobbies and interests and life itself were suddenly seen and approached in a different light. Science was the answer to all questions. (Behavior of politicians excepted!)

From my first Microbiology class, bacteria fascinated me. As if they were people, I was introduced to these microbial characters one by one and began to realized they too had quirks and personalities. They could be grouped and identified. Some were good, some not so much. Like people, they liked to live in different locations and each preferred to “eat” different foods. “Family Trees” could show how they were related to each other. Many were industrious workers which could be harnessed to produce products we need or destroy those we don’t. As in society, some were parasites which preferred to live off others. Microbes could themselves catch infections and become “sick”. Bacteria were “intelligent” in the sense that they could detect and respond to adverse environmental conditions, such as antibiotics, and adapt accordingly. They were survivors. Microbes were not just static “Germs” but rather were ever evolving complex life forms.

After obtaining my honours degree in Microbiology & Immunology and a certificate in Medical Laboratory Technology, I spent over thirty years employed by various medical institutions. At times I have been involved in teaching, research and the day to day diagnosis of medically important infectious diseases. I have found it rewarding passing on knowledge of this fascinating discipline to students, managed to co-author some medical papers based on my research, and had the tremendous satisfaction of diagnosing the day to day infectious diseases afflicting ailing patients. My last 20 years have been spent as a Microbiology Laboratory Technologist at a local acute care community hospital.

Whether your doctors suspects infection by bacteria, fungi, viruses or parasites, your specimens will be examined by a highly trained and dedicated Medical Laboratory Technologist, registered with the Collage of Medical and Laboratory Technologists of Ontario (CMLTO). Medical technologists occupy one corner of the ‘medical health triangle’ (1)

It is rather ironic that it was a bacterium that ultimately found revenge and caused an infection on my spine leaving me a paraplegic. Bacteria are all around us. We become colonized by bacteria from the environments we reside in. A hot soapy shower may reduce the numbers temporarily however in as little as twenty minutes they will start to emerge from where they were hiding and re-colonize your body. It is a fact of life! No doubt my day to day presence in a hospital setting during the last 30 years had me carrying bacterial strains which could be traced back to my workplace. Regardless, my infection was not directly work related but probably resulted from my run down state after a 10 day stretch at work. I developed a sprained back with familiar symptoms and thought that bed rest might help. Not finding anyone willing to take over his established medical practice, I found myself without a family physician shortly before my symptoms developed. I opted for a trip to a local walk in clinic rather than tolerate a painful six hour wait in my hospital’s crowded emergency department. Muscle relaxants and pain killers were prescribed as I crawled my way back to bed and sank into a hazy semi-conscious state. Too painful to shower, I survived in a sweaty incontinent state unwilling to eat while continuing to deteriorate. Perhaps it was through my pneumonia, a urinary tract infection, or I may have inoculated myself with resident bacteria through a scratch. Regardless, I emerged from my state of oblivion long enough to realize that my legs would not move. My wife called an ambulance and as my hospital’s emergency department continued to exist in an overcrowded state, I was diverted to another community’s hospital. After numerous tests, it was determined that I had a urinary tract infection (UTI) which was probably the source of my blood culture infection and state of septicaemia (2). An MRI (3) ultimately revealed that I had an abscess on my spinal column. The culprit was an old friend of mine - Staphylococcus aureus. As serious at this situation was, the bacterium was not the highly resistant MRSA (4) strain and so could be treated with less toxic antibiotics.

A course of antibiotic therapy was initiated but did not clear the abscess, therefore surgery was required to excise and drain the abscess. Post surgical antibiotic therapy was administered for 6 additional weeks as intravenous Oxacillin to ensure complete eradication of my newly acquired companion. Sadly neither surgery nor antibiotic therapy could resolve my paraplegic condition.

A veterinarian that has been bitten by a dog would hardly give up his practice nor develop a hate of canines. Likewise, I continue to love bacteria and hope to someday get back to my laboratory and my fascinating microscopic friends.

Staphylococcus aureus: (A Most Basic Primer)

Staphylococcus aureus is a coccoid (5) bacterium which divides in two planes giving it a “grape-like” clustered appearance under the microscope. A biological stain known as the “Gram Stain” colours them purple.

Cell wall components determine if a particular bacterium is either Gram Positive (Purple) or Gram Negative (Red). This initial differentiation of bacteria provides information as to how to proceed further for identification and a rough idea of what antibiotics could be excluded from consideration.

Specimens are plated (6) on appropriate bacteriological media (think food) and allowed to grow up from microscopic cells to visible colonies comprised of millions of cells. Macroscopic appearance and growth patterns on various media provide further clues.

The Genus Staphylococcus contains numerous species however Staph aureus is probably the most medically significant. Staph aureus can appear a golden-yellow colour when grown on Sheep Blood Agar whereas the less invasive Staph epidermidis appears as white colonies.
Staph aureus can also produce enzymes called haemolysins which, as the name implies, lyses the blood causing a clear zone around each colony.

Picking the colonies with a wire or loop, they can be tested by various methods or inoculated into a variety of media to see what enzymes they possess and what products they use or produce. The patterns obtained help to differentiate a particular bacterium from other species as well as other genera.

Basic Biochemical Traits of Staph aureus:

Beta-haemolytic - enzymes present lyse the blood cells clearing the area around the colonies.
Catalase Positive - Staph species poses enzymes which can split 3% hydrogen peroxide into oxygen and water to give observable bubbling when colonies are submersed in it.
Coagulase - Staph aureus produces an extra cellular protein which causes plasma to produce visible clumps when mixed together with the bacterial cells. Staph epidermidis does not
DNAse - Staph aureus can produce and enzyme which degrades DNA. When incorporated into a growth medium with an indicator the degradation of the DNA can be detected if present. Other Staph do not produce DNAse but some other genera do.
Mannitol Fermentation - Staph aureus can ferment this particular sugar where Staph epidermidis cannot. Resulting acid production from the fermentation can be detected by colour indicators.
Salt Tolerance - Staph aureus has a particularly high tollerance to salt concentrations. In the lab they can be routinely tested against 6% sodium chloride (NaCl or salt) but can often tolerate up to 15% NaCl.
Facultative Anaerobe - Staphylococci species are “Facultative Anaerobes” meaning they like to grow in the presence of air or oxygen (aerobic) but can survive nicely in (anaerobic) environments or those deprived of oxygen.

In addition to these products, Staph aureus can produce a myriad of enzymes, proteins and factors which can aid the microbiologist in identification and aid the bacterium in defeating host defence mechanisms thereby assisting it in establishing infections. Some of these are Staphylokinase, Leukocidin and Exfoliatin toxin.

Staph aureus can exist as normal flora on healthy people. They are often found in the nasal passages and the rectal area. Staph aureus is considered to be opportunistic in that it can become a pathogen under certain conditions.

Illnesses Produced by Staphylococcus aureus:

Boils and Pimples (folliculitis)
Pneumonia
Food Poisoning
Septicemia (Blood Poisoning)
Toxic Shock Syndrome
Osteomyelitis (Invasion of the bone)
Surgical Wound Infections
Scalded Skin Syndrome

Historically Staphylococci were sensitive to penicillin however over the years, antibiotic pressure selected for those rare bacterial cells that could produce an enzyme capable of degrading penicillin. Now virtually all Staph aureus strains are resistant to penicillin and should be considered as such. Modified penicillin derivatives were created which were resistant to these enzymes. Hospital acquired infections are often caused by the antibiotic resistant strain (MRSA) and can only be treated with vancomycin or a newer alternative. Vancomycin is considered to be the last best antibiotics in the arsenal against Staph aureus. VRE or vancomycin resistant strains are now showing up leaving few alternatives for treatment. Until recently, infections acquired outside hospitals have been treated with penicillinase-resistant ß-lactams. However, many of the community acquired (CA) Staphylococcal infections are now methicillin resistant. These organisms are uniformly resistant to penicillins and cephalosporins. The infections have been treated with combination therapy using sulfa drugs and minocycline or rifampin.

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So now I, the practitioner who would on any other day diagnose this sort of infection, now became the patient and had to put my diagnosis in the hands of my colleagues. They did not disappoint.

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(1) The Medical Health Triangle - Three components compose what is known as the Medical Health Triangle with Doctors at the pinnacle. The Doctors are supported by a base of Nurses and Technologists.
Many specialties and disciplines are found within each group. As Technologists usually would have the least patient contact, their contribution to a patient’s health is under appreciated.

(2) Septicaemia - A condition where bacteria have invaded and are actively growing in the blood stream.

(3) MRI - Magnetic Resonance Imaging: a non-invasive method used to create images of physiological and pathological features within the body. Principles of action are beyond the scope of this post.

(4) MRSA - Methacillin Resistant Staphylococcus aureus - an acronym for this particular strain of bacteria which can defeat the action of the antibiotic of choice, methacillin/oxacillin. Greatly reduces the choices of antibiotics which can be used to cure this infection.

(5) Coccoid: Bacteria can exhibit a variety of shapes and forms. The two most common forms are “coccoid” round spheres (coccus) or elongated rods (bacilli).

Gram Positive Cocccus vs. Gram Negative Bacillus

(6) Plating - Specimens suspected of being infected with bacteria are “plated” or inoculated onto media which can assist in their identification. Media can serve several purposes:
a) Growth Media - encourages the growth of any bacteria present.
b) Selective Media - which will encourage the growth of suspect bacteria while inhibiting bacteria which are unimportant to the disease process.
c) Differential Media - have components such as indicators which allow the identification or detection of certain bacteria by their metabolic properties.

Staphylococcus aureus = Staph aureus = S. aureus

Further reading on Staphylococcus aureus

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