Sure, put tuberculin in everyone’s eyes.

If you’ve worked in a health care facility, you’ve probably been given the tuberculin skin test. You get a little injection under the top layer of your skin, forming a bubble, and an allergic reaction means you’ve been infected in the past by the tubercle bacillus, or Mycobacterium tuberculosis as we now know it. If you haven’t been infected in the past, you’ll have slight discoloration and maybe slight pain.

Or it may mean you’ve been infected by another species of Mycobacterium. There’s a separate skin test for Mycobacterium avium complex, the “MAC infection” that’s becoming more common, but cases of M. avium often turn up positive from the M. tuberculosis test as well. The material used for the test consists of a purified solution of protein (PPD, or purified protein derivative) extracted from the bacteria.

* * *

The tuberculin skin test is also known as the Mantoux test, and has been for over a century, since Mantoux’s practical application of the hypersensitivity reaction discovered by von Pirquet. There were alternatives for much of that time, all variations on the theme of a small skin injection. The Heaf test, for example, was easier to administer consistently, and probably easier to interpret, but harder to manufacture.

And there were more unusual alternatives, early in the 20th century.

In 1908 three Philadelphia physicians, Samuel McClintock Hamill, Howard C. Carpenter and Thomas A. Cope reported the results of comparisons of several diagnostic tests for tuberculosis. These tests involved administration of tuberculin to different sites in the body: conjuctiva (Calmette); deep muscle (Moro); and skin (von Pirquet).

(from “Orphans as guinea pigs: American children and medical experimenters, 1890-1930” by Susan E. Lederer)

Conjunctiva? That’s… the eye, right? They put tuberculin in the eye, creating an irritation at worst, and a major allergic reaction and possible scar tissue if the test was positive? This was done to people, just as a screening test?

Indeed. Remember, back then a simple injection was not as trivial as it is now. Needles and syringes were not disposable, so the Pirquet test involved scarifying the skin and applying tuberculin into the wound. And if a routine injection led to a hospital-acquired infection, there were no antibiotics to treat it. Dropping some liquid in the eye was easier. More from Lederer’s monograph:

 The physicians explained that before beginning the conjunctival test, they were unacquainted with any adverse effects associated with the procedure. The ease of implementing the test (application of a few drops of tuberculin to the surface of the eye) and the relatively quicker results obtained thereby made it attractive to clinicians in search of an effective diagnostic tool. However, in the course of testing, several disadvantages quickly became manifest. The reaction produced a ‘decidedly uncomfortable lesion’ and in several cases, serious inflammations of the eye resulted. In addition, the possibility that permanent impairment of vision might result for several children worried the physicians.

The test proved useful, revealing that many of the children had had undiagnosed cases of tuberculosis. But it was unpopular.

from the Reading Eagle newspaper, 1910

* * *

What were the arguments for and against the eye test?

In the Journal of the Missouri State Medical Association (November 1908), L. M. Warfield explains that the skin test is more sensitive, as it gives positive results from people who have already recovered from tuberculosis, or who show no signs of disease.

This goes along with his instinct for which one is safer: “I have used the cutaneous reaction more than the ocular reaction, for the eye is too delicate an organ to be played with.”

Another complaint about Calmette’s ocular test is that it should not be done on eyes that are suffering any other malady, which is hard to guarantee. In the New England Journal of Medicine (August 27, 1908) Dr. Egbert LeFevre illustrates how complications may arise.

* * *

Within the first year of its introduction the eye test for tuberculosis was already losing fans.

In February 1908, an article by Floyd and Hawes saw the eye test as safer than the skin test — they could be summarized to say “the advantages of the ophthalmo-tuberculin reaction over the cutaneous or subcutaneous methods is that it is absolutely painless, whereas both of the others are painful or disagreeable to say the least. Practically no constitutional symptoms follow the use of the eye, whereas in the subcutaneous test they are important to obtain and often very distressing, and also occasionally occur in the cutaneous method.”

Six months later, doctors were abandoning the procedure. T. Harrison Butler of Coventry, England laid out the empirical observations that changed his mind in the August 8, 1908 British Medical Journal.

Further argument against the eye test came from L. Emmett Holt of New York, whose paper in the January 1909 Archives of Pediatrics (along with the Philadelphia one mentioned above) became a massive controversy when publicized by “anti-vivisection” activists. The title is a bit alarming. (“Babies Hospital” is now called Children’s Hospital of New York-Presbyterian.)

According to Holt, not only does the eye test produce unnecessary discomfort, it’s actually harder to perform.

In ease of application there is a decided advantage in the skin test. The scarification is a trifling thing. The patient does not require continuous observation before or after, and the reaction lasts a considerable time. The ophthalmic cases need closer watching, the reaction is shorter and may be missed. It cannot be used well in ambulatory patients.

The 1909 Eye, Ear, Nose and Throat annual points out yet another practical limitation.

Still optimistic about the eye test, the New York State Journal of Medicine blames problems on improper technique.

In considering the ophthalmic test we must call attention to the fact that harmful results are in all probability due to the instillation of tuberculin into diseased eyes, to infection after instillation, or mechanical irritation, to the introduction of secretion by the fingers of careless patients into the untested eye and to the use of poor or faultily prepared tuberculin.

Calmette reports 13,000 instillations and states that in no case in which the tests were properly applied and controlled were there serious complications. Petit tabulated 2,974 instillations with no ill effects in 698 positive reactions. Smithies and Walker in 450 instillations in 377 patients had four stubborn reactions. It is wise to remind the profession that the eye needs to be thoroughly examined before the test is made and with the slightest abnormality, tuberculin should not be used.

It’s agreed that the test shouldn’t be given to people with any eye problems, and it can’t be given more than once on the same eye (in a lifetime?), and it shouldn’t be given to old people. And maybe you should keep some cocaine around to numb the eyes of children and “sensitive adults” so they don’t squeeze the irritant out of their eyes.

With all these limitations, you’ll have to learn how to use the skin test anyway. So you might as well use it all the time. By 1911 Theodore Potter of Indiana University writes that “the eye reaction has already largely fallen into disuse, being replaced by the von Pirquet test.”

The eye test is still good for cattle, though!

 

 

 

Pubmedwhack: Immuno-DTO

Today’s Pubmedwhack comes from the world of unstable metals and electron microscopy. For the definition of “Pubmedwhack”, see this earlier post.

* * *

Following the invention of immunofluorescence, scientists developed other methods for using labeled antibodies to identify certain proteins or substances under a microscope.

There’s immunohistochemistry (IHC), in which the antibodies are labeled not with a fluorescent marker, but with an enzyme which produces a visible reaction in the presence of a substrate. This is less precise, but lets you see your protein of choice under visible light while also looking at the structure of the tissue. The slides labeled this way last longer instead of being quenched by the microscope’s light source.

You can also use labeled antibodies to see things under the electron microscope (immuno-electron microscopy or immuno-EM). In 1960 Rifkin et al. (1) published an image of virus particles on a cell surface, labeled with ferritin-conjugated antibodies. As you can see, instead of the labeled antibodies changing the color of a region of the cell, each individual labeled antibody is visible as a “granule”.

from Rifkin et al (1960), Nature 187:1094

This was made possible by an innovation published one year earlier, entitled Preparation of an electron-dense antibody conjugate (2). Ferritin is a small protein which just about all organisms use as an iron carrier. When “iron-loaded”, almost a quarter of its mass is iron atoms. Therefore this is an especially electron-dense molecule, visible as a dark spot under the electron microscope, as seen above. Soon, further advances let scientists see ferritin-labeled structures inside cells.

* * *

For about a decade ferritin was the label of choice for immuno-electron microscopy. Then in 1971, a new technique came along (3, 4), in which antibodies were mixed with a solution of colloidal gold until they absorbed to the metal’s surface. Gold-labeled antibodies could be separated from free antibodies by centrifugation. Immunogold is still the dominant immuno-EM staining method 40 years later.

from Faulk et al (1971), Nature New Biology 231:101

* * *

In the 1960s, other techniques were created for immuno-EM. I have almost no EM experience and don’t know the pros and cons, but clearly there was a desire to get rid of the protein element of the electron-dense antibody label, and just attach the antibody to a metal ion. The protein was unnecessarily big, and subject to denaturation. So the 1960s also saw a lot of papers using antibodies labeled with mercury (technically the diazonium salt of tetraacetoxymercuriarsanilic acid (5) and p-(aminophenyl)-mercuric acetate (6), phrases which mean little to me).

from Zhdanov et al (1965), J Histochem Cytochem 13:684

There were also studies using antibodies labeled with heavier metals. In his long career, Ludwig Sternberger and his lab (at Johns Hopkins and elsewhere) invented several microscopy techniques, with the most important probably being the horseradish peroxidase (see original paper (7), and appreciation of it as a “citation classic”).

He also spent much of the 1960s devising improved metal-based antibody labels for electron microscopy, including immunouranium (8), immunouranium with added osmium for enhanced contrast (9), and finally immuno-diazothioether-osmium tetroxide (10), or immuno-DTO. The uranium methods seem somewhat useful, but as far as I can tell were only used by Sternberger’s own lab. Immuno-DTO in particular seemed almost unusable; they used it more than once, but a Pubmed search for “Immuno-DTO” only returns one result (11). As Sternberger himself says in a review (12) of his and other techniques:

Unfortunately, the diazotized diazothioethers are not very stable even at Dry Ice temperatures and the solid deteriorates in a few days. Therefore, it was not surprising to observe that the osmnium tetroxide-binding iower of diazothioether antibodies was unstable, even on storage in liquid nitrogen.

Oh well, it was a nice idea.

* * *

1. Rifkind RA, Hsu KC, Morgan C, Seegal BC, Knox AW, Rose HM (1960). Use of Ferritin-Conjugated Antibody to Localize Antigen by Electron Microscopy. Nature 187:1094-1095.

2. Singer SJ (1959). Preparation of an Electron-dense Antibody Conjugate. Nature 183:1523-1524.

3.Faulk WP, Taylor GM (1971). An Immunocolloid Method for the Electron Microscopy. Immunochemistry 8(11):1081-1083.

4. Faulk WP, Vyas GN, Phillips CA, Fudenberg HH, Chism K (1971). Passive Haemagglutination Test for Anti-rhinovirus Antibodies. Nature New Biology 231:101-104.

5. Pepe FA (1961). The Use of Specific Antibody in Electron Microscopy: I: Preparation of Mercury-Labeled Antibody. J Biophys Biochem Cytol 11(3):515-520.

6. Zhdanov VM, Azadova NB, Kulberg AY (1965). The Use of Antibody Labeled with an Organic Mercury Compound in Electron Microscopy. J Histochem Cytochem 13(8):694-687.

7. Sternberger LA, Hardy PH Jr, Cuculis JJ, Meyer HG (1970). The Unlabeled Antibody Enzyme Method of Immunohistochemistry: Preparation and Properties of Soluble Antigen-Antibody Complex (Horseradish Peroxidase-Antihorseradish Peroxidase) and its Use in Identification of Spirochetes. J Histochem Cytochem 18(5):315-333.

8. Donati EJ, Figge FHJ, Sternberger LA (1965). Staining of Vaccinia Antigen by Immunouranium Technique. Exp Mol Pathol 4(1):126-129.

9. Sternberger LA, Hanker JS, Donati EJ, Petrali JP, Seligman AM (1966). Method for Enhancement of Electron Microscopic Visualization of Embedded Antigen by Bridging Osmium to Uranium Antibody with Thiocarbohydrazide. J Histochem Cytochem 14(10):711-718.

10. Donati EJ, Petrali JP, Sternberger LA (1966). Formation of Vaccinia Antigen Studied by Immunouranium and Immuno-diaxothioether-osmium tetroxide Techniques. Exp Mol Pathol Apr:Suppl 3:59-74.

11. Sternberger LA, Donati EJ, Hanker JS, Seligman AM (1966). Immuno-diazothioether-osmium tetroxide (immuno-DTO) technique for staining embedded antigen in electron microscopy. Exp Mol Pathol Apr:Suppl 3:36-43.

12. Sternberger LA (1967). Electron Microscopic Immunocytochemistry: A Review. J Histochem Cytochem 15(3):139-159.

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.

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.

* * *

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.

* * *

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.

* * *

(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)

—

(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)

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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)

—

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)

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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)

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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)

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(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)

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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)

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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)

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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)

—

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)

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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)

What’s a “full-sized drop” in nanoliters?

A lot has been made nowadays about reproducibility, and how for some reason the most interesting scientific results are often the least reproducible. Evidently when there’s huge pressure to generate a certain graph containing certain data, either to give yourself a chance at fame and fortune or to give yourself a chance to have your lab and scientific career continue to exist, sometimes the graph does not represent the sort of objective reality that exists throughout space and time. This can be because of wishful thinking, because of selective use of the data that seems most solid, because of variables we never considered which later turn out to be crucial, who knows what else.

There have always been experiments where different labs get different results. Often we say that “in our hands,” we get Result X, but another laboratory gets Result Y, and we can say this without accusing anyone of malfeasance. It’s acceptable to get different results.

But nowadays, a scientist has no excuse when his contemporaries can’t even figure out how to replicate an experiment. A hundred years ago, it was more tricky. Even if you wrote and asked for a detailed protocol, it would probably involve terms that had no exact meaning. The topic under discussion today is the word “drop”.

* * *

In the December 1910 issue of the JAMA organ Archives of Internal Medicine, Drs. C. C. Bass and John A. Watkins wrote up (1) a new quick-and-easy test for typhoid that they had developed in the laboratories of Tulane University. The abstract is here, with subscription required to read the article. But this volume is old enough that it’s out of copyright and should be in Google Books. Though to be honest I can’t find it there and only found it at archive.org.

Over the ensuing years many doctors’ offices used it with more or less success, but as you might expect, many found they were unable to get results and went back to their old routine of sending samples to a clinical lab and waiting a day or two.Even though Bass and Watkins went to the trouble of including highly mundane photographs of things like proper slide-rocking procedure, people couldn’t figure out what exactly they were supposed to do. The text still contains phrases like “two to three drops of an equal number of bacilli units and agglutinin units sufficiently dilute to prevent rapid agglutination”. And “this one-quarter drop of blood is about the quantity we use in making blood slides in examinations for malaria, differential counts, etc.” The instructions are easy to understand, but really to communicate this sort of information you have to show people and let them practice it.

As a result, the 1910s saw some skeptical questions came in to the miscellaneous letters section of JAMA, sometimes with fairly impatient replies from the editors.

The editors of JAMA explain in detail the benefits of the Bass-Watkins test. (citation #2)

Following the usual routine of randomly skimming randomly selected old journals, I found a follow-up piece in the July 1918 New Orleans Medical and Surgical Journal (3) which goes into further detail trying to delineate concepts like “drop”. The author (presenter, rather, since this is the transcript of a talk which was followed by comments) is one of Bass and Watkins’s junior colleagues at Tulane, Foster M. Johns. Here’s what he says.

In the eight years that have elapsed since the publication of this article, this reaction has constantly grown in favor of the clinicians of the South, in spite of many improper lots of reagent supplied by private laboratories, my own included, as well as the various biological houses. During this time the test has been in constant use in the laboratories of clinical medicine with which I am connected, and it is with the belief that this reaction offers an easier, quicker and even more accurate reaction to not only the clinicians, but the trained laboratory worker as well, that I have prepared this discussion of a well-known test. During this time the few faults in technic and production brought out by continual use have been met and overcome, with the exception of a technic that will insure the uniform production of a stock suspension of typhoid bacilli that will keep well under the ordinary conventions of usage.

…

As simple as the technic sounds, there is often considerable difficulty in doing a simple thing. Taking up the test step by step, I will endeavor to point out the places where error may creep in. To begin with, an absolutely clean slide, freshly washed with soap and water to remove the grease and dust, most be used. Now, we require one-quarter of a drop of blood on the center of the slide. This is a quantity almost impossible to describe to one not accustomed to the routine making of proper blood smears, but practically it is easily approximated. Squeeze a quantity of blood out of a puncture on the finger or ear lobe that will not quite drop off, and then barely touch the slide to it. The quantity adhering to the slide will vary from one-quarter up to one-half of one drop. In either instance, for practical purposes, the end result will not be influenced… The actual dilution of the organisms will not be disturbed by either of the quantities of blood, as the blood is then spread roughly over the middle third of the slide and allowed to dry.

Now, one drop of plain water is added. Drops can vary enormously in size, and while, if the proportions in the test were carried out to suit, no harm would ensue, still, for working purposes, we need a full-sized drop. In this instance the standard drop is measured by preferably using the ordinary medicine dropper held almost parallel to the table, so that the drop collects on the side of the elongated glass tip of the dropper.

Full-sized drop? Quarter drop? A quarter drop is not a drop divided into fourths, but a drop that will not quite drop off? Or do you really mean a “finger or ear lobe that will not quite drop off”? In which case the real concern may be not typhoid, but leprosy.

Instead of all this … wouldn’t it be easier to measure volume in microliters?

I know nothing about the history of scientific equipment, but Wikipedia reports that there were no micropipettes until 1960.  I don’t think there were syringes capable of measuring volumes on the level of a drop, either. And if there were such devices, they were far from disposable, and would need to be cleaned and dried between uses.

* * *

What is a drop anyway? And what was the smallest amount of volume that could be accurately measured a hundred years ago?

Again to the Wikipedia, which claims that today there is a medical definition of “drop” as 50 microliters. That means a quarter drop is 12.5 microliters, which sounds like about the right amount for a blood smear that you would quickly look at under a microscope.

Apothecaries traditionally were able to make much more precise measurements of weight than of volume. The common measurement of a “grain” is equal to 1/20 of a scruple, or 1/60 of a dram. And a dram is only 1/8 of an ounce, so there are 480 grains in an ounce, making a grain about 64 milligrams, under the old system where there were 12 ounces in a pound and a pound was about 1/3 of today’s kilogram.

Meanwhile, for liquids, people didn’t have too much trouble measuring in terms of scruples (slightly more than a cc or milliliter). But the minim, the volumetric equivalent of the grain, was only invented around the beginning of the 19th century, and required quite specialized equipment. Being the equivalent of a grain, the minim is about 64 microliters, or roughly… a drop. So it just made sense to refer to things in terms of drops. But when you factor in surface tension, temperature, the size of the vessel from which the drop is dropping… it’s always a judgment call. As the old saying goes, blood has a higher viscosity and specific density than water.

So those of us with access to space-age technology like micropipettes (and disposable anything) should count our blessings.

* * *

1. Bass CC, Watkins AA (1910). A quick macroscopic typhoid agglutination test. Arch Inter Med VI(6):717-729.

2. from “Miscellany”, September 5th (1914). Value of von Pirquet reaction in adults / Reliability of Bass-Watkins test. J Am Med Assoc LXIII(10):883.

3. Johns FM (1918). The Bass-Watkins agglutination test for typhoid. New Orleans Med Surg J LXXI(1):22-27.