Wednesday, 30 June 2010

Aspergillus species Foot Cell

Aspergillus (species in general)

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;
Volume 49, Numbers 2-3, 209-215,
Ratna Singh

Tuesday, 29 June 2010

Quellung Reaction (Streptococcus pneumoniae)

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.

Return Home (Most Recent Posts)

Rhizopus species


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.

Nocardia species

(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

Return Home

Sunday, 13 June 2010

Aspergillus niger

(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)

Return to 'Home' -(most recent posts)

Enterobius vermicularis (Pinworm)

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.

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.

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)
  1. Lower left worm female showing ovaries containing developing pinworm eggs
  2. Central worm has ruptured wall
  3. All four worms have characteristic projections called 'alae' which on the worm furthest to the right I've highlighted with arrows.
  4. 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. Two 'alae' are also seen at bottom left-of-center and top right-of-center of worm. (X400)

New; February 2012
I decided to add these photos here rather than place them in a new post as I had not photos showing the entire worm and though this might further add to the subject.

These pinworm specimens were obtained not from a adhesive tape prep from around the anus but rather taken during a colonoscopy on an adult male. The pinworm generally makes it's home in the large intestine and cecum.

Female Pinworm showing buccal cavity, esophagus and median bulb.
(Inset is the pointed tails of the female worm (thus the name 'pin'worm)
Nikon Camera 250X -(click on any photo to enlarge for better viewing)

As Above; Female with Buccal Cavity, Esophagus and Median Bulb visible.
Wet Preparation direct from colonoscopy material: (DMD-108 Microscope X250)

Female Pinworm (Entire) (Micron Scale Bar in upper right corner of photo)
Worm measured 2.65 cm in length & 180 µm at widest point of body
(Taken with DMD-108 Microscope X100)

Male Pinworm distinguished by it's curved caudal end/tale not all that convincing in this photo in which the worm is not straight but loops in the centre.
(DMD-108 Microscope X250)

Return Home

Alternaria alternata

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.

- 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.
- 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.

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)

Return Home

Friday, 11 June 2010

Whipworm (Trichuris trichiura)

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.

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.

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

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)

Return Home

Giardia lamblia

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.

- 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 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.

- 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)

Return Home Most Recent Posts