Amboceptor

Microbiological, virological, bacteriological, immunological, medical, epidemiological, historical, anecdotal

Tag: Gallery

Gallery: The amazing ink-proof yeast capsule

Observed doctors and medical students as they learn about the workings of the clinical microbiology lab, I’m impressed by their love of the India ink test for cryptococcus. The way this test works is: Cryptococcus is a type of infectious yeast that looks a lot like Candida if you just do a gram stain. But it has a polysaccharide capsule around each cell (unless for some odd reason it isn’t producing a capsule), wider than the cell itself. So if you put Cryptococcus in a colored liquid, most famously a solution of India ink, the polysaccharide capsule shows up as a huge empty white area around the cell. Whereas with Candida, only the cell itself is white.

We apparently don’t use this test regularly anymore, but we still show it to people in case they need to know what it is.

Something about the India ink test just makes people happy. A lot of diagnostic microbiology uses techniques that were developed several generations ago, but this one is just so simple, requiring not “acid alcohol” or various toxic red and purple substances, but merely the simplest form of ink, developed millennia ago. And to use the phrase “India ink”, instead of “colloidal carbon” or something, is such an anachronism in the 21st century. Most of us last saw that phrase when reading some classic of literature like The Secret Garden or A Bear Called Paddington. And aside from the name, there’s something magical about seeing this invisible capsule appear around what seemed to be a normal yeast cell. Like lemon-juice ink made visible.

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So here are some depictions of India-ink-stained Cryptococcus in the literature. First, camera lucida drawings from a 1935 JID paper by Rhoda W. Benham (Cryptococci — their identification by morphology and serology) that must have been a handy field guide to Cryptococcus species. The top right corners of the dishes are shaded to show how they look under India ink.

crypto-benham

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Now, some photos of patient tissues directly stained with India ink.

From Wilson HM, Duryea AW (1951), Cryptococcus meningitus (Torulosis) treated with a new antibiotic, actidione®. Archives of Neurology & Psychiatry 66(4):470-480.

crypto-duryea

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From Carnecchia BM, Kurtzke JM (1960), Fatal toxic reaction to amphotericin B in cryptococcal meningo-encephalitis. Annals of Internal Medicine 53(5):1027-1036.

crypto-carnecchia

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From Schupbach CJ, Wheeler CE Jr, Briggaman RA, Warner NA, Kanof EP (1976), Cutaneous manifestations of disseminated Cryptococcosis. Archives of Dermatology 112(12):1734-1740. Note “Tzanck preparation”, looking for multinucleated giant cells.

crypto-schupbach

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From Love GL, Boyd GD, Greer DL (1985), Large Cryptococcus neoformans isolated from brain abscess. Journal of Clinical Microbiology 22(6):1068-1070.

crypto-love-1985

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From Bottone EJ, Kirschner PA, Salkin IF (1986), Isolation of highly encapsulated Cryptococcus neoformans serotype B from a patient in New York City. Journal of Clinical Microbiology 23(1):186-188.

crypto-bottone

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And some images of cells grown in culture. Ending with one in color!

From Neill JM, Abrahams I, Kapros CE (1950), A comparison of the immunogenicity of weakly encapsulated and of strongly encapsulated strains of Cryptococcus neoformans (Torula histolytica). Journal of Bacteriology 59(2):263-275.

crypto-neill

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From Littman ML, Tsubura E (1959), Effect of degree of encapsulation upon virulence of Cryptococcus neoformans. Proceedings of the Society for Experimental Biology & Medicine 101:773-777.

crypto-littman

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From Bulmer GS, Sans MD, Gunn DM (1967), Cryptococcus neoformans I: Nonencapsulated mutants. Journal of Bacteriology 94(5):1475-1479.

crypto-bulmer

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From Dykstra MA, Friedman L, Murphy JW (1977), Capsule size of Cryptococcus neoformans: Control and relationship to virulence. Infection & Immunity 16(1):129-135.

crypto-dykstra

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From Chang YC, Kwon-Chung KJ (1994), Complementation of a capsule-deficient mutation of Cryptococcus neoformans restores its virulence. Molecular & Cellular Biology 14(7):4912-4919.

crypto-chang

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From Doering TL (2000), How does Cryptococcus get its coat? Trends in Microbiology 8(12):547-553.

crypto-doering

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From Zaragoza O, Casadevall A (2004), Experimental modulation of capsule size in Cryptococcus neoformans. Biological Procedures Online 6(10):10-15.

crypto-zaragoza-2d

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From Zerpa R, Huicho L, Guillén A (1996), Modified India ink preparation for Cryptococcus neoformans in cerebrospinal fluid specimens. Journal of Clinical Microbiology 34(9):2290-2291.

crypto-zerpa

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And a bonus: High-tech 3-dimensional visualization! These are 40 focal “slices” of a single cell. From Zaragoza O, McClelland EE, Telzak A, Casadevall A (2006), Equatorial ring-like channels in the Cryptococcus neoformans polysaccharide capsule.

crypto-zaragoza-3d

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Gallery: Early immunofluorescence

How did immunofluorescence begin? Who was the first scientist to use fluorescently labeled antibodies to stain cells under a microscope?

The answer to this is pretty clear. Albert Coons of Harvard Medical School, in two papers from 1942 and 1950. In this 1962 speech (published in JAMA) Wesley Spink of the University of Minnesota describes Coons’s accomplishments.

The first time fluorescent molecules were attached to antibodies was in the early 1930s, as described in a brief letter to Nature by John Marrack (1934). But at this point the antibodies are being used in suspension, for serological experiments similar to those that detect bacteria by agglutination. In this case the bacteria can be measured colorimetrically by mixing them with fluorescently labeled antibody, washing off nonspecific antibodies, and seeing if the bacteria turn pink.

In fact, here’s the entire article.

marrack-1934-nature

In The Demonstration of Pneumococcal Antigen in Tissues by Means of Fluorescent Antibody (1942), Coons and associates showed that fluorescein isocyanate (FIC) could be attached to antibodies and used to stain thin tissue sections on slides, in the same way many histological stains had been used in the past. Rabbits were immunized with pneumococcus, and their serum was conjugated with FIC. This was used to visualize the bacteria in the liver of a mouse with severe pneumococcus infection. The first immunofluorescence image ever published, I believe, was this 20-minute (!) exposure.

coons-1942-fluorescence

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But the fluorescent antibody technique didn’t take off until Coons and Kaplan published the sequel eight years later. I can’t find any papers from the 1940s that cite the Coons paper and actually include fluorescent images of their own.

The pivotal 1950 paper is entitled Localization of Antigen in Tissue Cells: II: Improvements in a Method for the Detection of Antigen by Means of Fluorescent Antibody. Kind of like Rambo III, the numbering of this title is odd because there was no Localization of Antigen in Tissue Cells Part I. Instead, it’s explained that “the first paper in this series was entitled ‘The Demonstration of Pneumococcal Antigen in Tissues by Means of Fluorescent Antibody'”, meaning that this is a sequel 8 years in the making.

And what a sequel! Over 2,100 citations, according to Google Scholar’s notoriously inflated algorithm. Meanwhile PubMed’s claim of 301 is a clear underestimate. It was cited a lot.

Suddenly everyone was able to use fluorescent antibodies to find out where their antigen of choice was located inside the cell, or inside the tissue, or inside the animal. Especially after 1958 when Riggs and colleagues at the University of Kansas showed how to make FITC (fluorescein isothiocyanate) conjugates, which are more stable than FIC and don’t require phosgene for their chemical synthesis. Using thiophosgene is no picnic either, but apparently it’s less deadly.

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Just as early issues of the Journal of Virology are filled with papers that show “The Ultrastructure of [Name of virus]” by taking random pictures of it with an electron microscope, it seems like it was easy to get a microscopy paper published in the early 1960s. You injected rabbits with a substance, labeled their serum with fluorescein, stained some slides, and put together a manuscript called “Demonstration of [Name of substance] in [Name of tissue] by the Fluorescent Antibody Technique”.

I decided to look through some papers that cite Coons and Kaplan (1950), to find examples of early immunofluorescence. Here are a dozen images, all from at least fifty years ago.

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finck-chicken-myosin-1956

(2) Chicken muscle fiber, stained with rabbit globulin specific for myosin (0.5 microns, 1000x). (3) A “dark medium phase contrast” image of the same slide for comparison. (Finck et al., J Biophys Biochem Cytol 1956)

shope-papilloma-virus-mellors-1960

(3) Cottontail rabbit papilloma, stained with rabbit serum specific for Shope papilloma virus and goat anti-rabbit (75x). (4) An H&E stain of the same slide, to show that the virus antigens are in the keratinized region. (Mellors, Cancer Res 1960)

avian-flu-breitenfeld-1957

Embryonic chicken fibroblast infected for 10 hours with the Rostock strain of fowl plague virus (now known to be influenza A virus H7N1), stained with rabbit serum specific for “g antigen” (now called NP) (950x). The same cell stained by Giemsa-Wright stain, to show the nucleus. (Breitenfeld and Schäfer, Virology 1957)

roberts-1957-rat-eye

Rat eye (lens and iris), stained with rabbit globulin specific for rat glomerulus (190x). Another section of the same eye stained with non-specific rabbit globulin. This is an example of the papers that used specific antiserum to find common antigens in seemingly unrelated tissues, in this case kidney and eye. (Roberts, Br J Ophthalmol 1957)

blood-groups-a-b-szulman-1960

Human tissues, stained with human serum specific for blood group A or B. Yes, human serum. A volunteer of blood group A was used to get the serum against blood group B, and vice versa. (Szulman, J Exp Med 1960)

GBS-Moody-1960

Group B streptococci, stained with rabbit globulin specific for group B streptococci (magnification unspecified). This paper’s total lack of negative controls is charmingly naive. (Moody et al., J Bacteriol 1958)

amoeba-brandt-1958

(5) Amoebae frozen during pinocytosis, with free fluorescent antibody (non-specific rabbit globulin) visible in pinocytosis vacuoles (2000x). (6) Phase-contrast view of another section of the same amoeba. (Brandt, Exp Cell Res 1958)

ferret-influenza-liu-1955

Mononuclear cells from nasal smears of ferrets infected with influenza (PR8 strain), stained with rabbit globulin specific for influenza virus (560x). Cells display a range of nuclear and cytoplasmic staining patterns. (Liu, J Exp Med 1955)

cladosporium-aldoory-1963

Cladosporium bantianum mold stained with rabbit serum specific for C. bantianum (1000x). By comparison, C. carrionii stained with serum specific for C. bantianum, to show lack of cross-reactivity. (Al-Doory and Gordon, J Bacteriol 1963)

yeast-kunz-1961

Mixture of two yeast species photographed under both ultraviolet and visible light simultaneously (magnification unspecified). Saccharomyces cerevisiae (near center) glows green when stained by rabbit serum specific for S. cerevisiae. Red light serves as counterstain for other species (Pichia membranefaciens). (Kunz and Klaushofer, Appl Microbiol 1961)

myoblast-holtzer-1957

A single myoblast isolated from a stage 23 chick embryo, stained with rabbit globulin specific for myosin (1100x). The arrow indicates where the nucleus obscures the myosin. (Holtzer et al., J Biophys Biochem Cytol 1957)

glomeruli-ortega-1956

Kidney of rat injected with nephrotoxic rabbit antibodies to cause nephritis; the rabbit antibodies concentrate in the glomeruli, as seen by staining with goat globulin specific for rabbit globulin (37x and 205x). (Ortega and Mellors, J Exp Med 1956)

Gallery: Cockroach feeding contraptions

Cockroaches are disgusting. The presence of roaches indicates squalid and germ-ridden conditions. However… do the roaches themselves contribute to the pestilence? Or are they merely a symptom? Or perhaps they are actually beneficial, as they might be able to destroy the germs in their digestive tract.

This is the sort of question that scientists needed to answer. Dr. Stanley Wedberg (1913-2003) of the University of Connecticut created an optimal system for such studies. The cockroach would be immobilized on a block of paraffin in what looks like a comfortable reclining position. This was suited for controlling its food intake to contain known amounts of bacteria, and monitoring its excretions to see how much bacteria remained.

Published in 1947, his system was based on one demonstrated a year earlier by Hubert Frings, a postdoc at Edgewood Arsenal in Maryland. What?

Yes, Edgewood Arsenal, now part of the Aberdeen Proving Grounds, was basically the US Army’s chemical plant, and included a Medical Research Laboratory, which in turn included a division of entomology and insect physiology for some reason. Hubert Frings’s mentor was the head of entomology, Dr. Leigh Chadwick.

Anyway, Frings (who was later quoted in Sports Illustrated on the subject of how to keep birds away from rocket sleds) was using fixed feeding rigs to get insects to ingest chemicals, to measure toxicity. In the paper that demonstrated the cockroach apparatus, his aims were even less sinister, basically looking at which salt solutions cockroaches prefer to drink, and figuring out how high of a concentration is necessary for roaches to prefer sucrose to water.

Wedberg improved the mechanism by putting the roach on its back, which reduced regurgitation and made it more comfortable (yes, both papers use the word “comfort” with regard to how the roach feels). And just as significantly, he decided to use a different roach species, one that was bigger (meaning food and excreta could be measured more accurately), more docile, and had a broad, flat body type well suited for attaching to a block of paraffin.

Wedberg’s choice of Blaberus cranifer as the experimental model cockroach did not catch on, but his technique did, and therefore many subsequent papers just referred to Wedberg and Clarke (1947) or Wedberg, Brandt and Helmboldt (1949) instead of including their own pictures of their own roach restraint systems. But several scientists did seek to show the reader what their apparatus looked like.

Here are a few such pictures. There are probably a lot more in the hardcore entomology journals (J Med Entomol, J Econ Entomol) whose archives I can’t access.

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From Frings H (1946), Gustatory thresholds for sucrose and electrolytes for the cockroach, Periplaneta americana (Linn.). J Exp Zool 102:23-50.

roach-frings-1946

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From Wedberg SE, Clarke NA (1947), A simple method for controlled experimentation on the passage of microorganisms through the digestive tract of insects. J Bacteriol 54:447-450.

roach-wedberg-1947

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From Mackerras IM, Pope P (1948), Experimental Salmonella infections in Australian cockroaches. Austral J Exp Biol Med Sci 26:465-470.

roach-mackerras-1948

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From Wedberg SE, Brandt CD, Helmboldt CF (1949), The passage of microorganisms through the digestive tract of Blaberus cranifer mounted under controlled conditions. J Bacteriol 58(5):573-578.

roach-wedberg-1949

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From Leibovitz A (1951), The cockroach, Periplaneta americana, as a vector of pathogenic organisms. I. The acid-fast organisms. Bulletin of the Pan-American Sanitary Bureau 30:30-41.

roach-leibovitz-1951

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From Julseth RM, Felix JK, Burkholder WE, Diebel RH (1969), Experimental transmission of Enterobacteriaceae by insects. I. Fate of Salmonella fed to the hide beetle Dermestes maculatus and a novel method for mounting insects. Appl Env Microbiol 17(5):710-713. [Well, they look like roaches. Small roaches.]

roach-julseth-1969

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From Klowden MJ, Greenberg B (1976), Salmonella in the American cockroach: Evaluation of vector potential through dosed feeding experiments. J Hyg, Camb 77(1):105-111.

roach-klowden-1976

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