Amboceptor

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

Month: January, 2014

What’s the deal with inverted typhoid?

Maybe it’s just lack of familiarity with other disciplines’ historical documents, but I feel like old medical and biological articles are particularly thick with phrases that are totally opaque now, but were self-evident to the people of the time. Phrases like “inverted typhoid” represent a type of jargon that is specific not just to a certain profession, but to a certain profession at a certain time in history.

* * *

First of all, what’s “typhoid”? Typhoid fever is a disease characterized by fever. There are other symptoms, like distended abdomen, diarrhea, elevated heart rate, “rose spots” on the abdomen, bacilli in the blood, and borborygmus. And positive results from molecular tests that range from the century-old Widal test for agglutinating antibody, to blood levels of liver enzymes. But the fever itself is a major part of the diagnosis.

This free chapter from 1990’s Clinical Methods (published by Butterworth, Boston) gives an overview of the different fevers caused by different diseases. Fever is typically lowest in the morning and highest in the evening, in a healthy person. Many sources say the typical range is between 0.5 and 1 degree Celsius, though this chapter says it can be as high as 1.5°C. Diagnoses based on various fevers can include:

Although not diagnostic, at times fever curves can be suggestive. Hectic fevers, because of wide swings in temperature, are often associated with chills and sweats. This pattern is thought to be very suggestive of an abscess or pyogenic infection such as pyelonephritis and ascending cholangitis, but may also be seen with tuberculosis, hypernephromas, lymphomas, and drug reactions.

Relapsing fevers may be seen in rat-bite fever, malaria, cholangitis, infections with Borrelia recurrentis, Hodgkin’s disease (Pel-Ebstein fever), and other neoplasms.

Historically, some diseases are described as having characteristic fever patterns. The double quotidian fever of gonococcal endocarditis has two spikes in a 24-hour period. Fever at 48-hour intervals suggests Plasmodium vivax or P. ovale; 72-hour intervals suggest P. malariae, while P. falciparum often has an unsynchronized intermittent fever.

So, you’ve got relapsing fever, hectic fever, double quotidian fever, and so on. What’s the pattern of typhoid fever? In his 1901 book Typhoid Fever and Typhus Fever, Heinrich Curschmann of the University of Leipzig gives some examples.

* * *

Here’s someone who came down with typhoid when he was recovering from “polyarthritis”, which is why they were already measuring his temperature. In all these graphs, the body temperature was measured once in the morning and once in the evening. Note that this guy starts out with small fluctuations between morning (low) and evening (high), and the difference between morning and evening gets exaggerated when the fever begins.

curschmann-fig8

Here’s the plateau stage of the fever, following the familiar “Wunderlich curve”. The difference between morning and evening continues to grow, even as the temperature itself levels off and gradually decreases.

curschmann-fig11

Here’s a guy who was thought to be faking illness but was brought to the hospital anyway. The point of this graph is that in his first week in the hospital he was not showing a particularly high temperature, but the large daily fluctuations should have been a warning sign that he might have typhoid.

curschmann-fig9

The “typhoid” pattern is a large fluctuation between morning temperature (low) and evening temperature (high). The period of low temperature can also be thought of as the “pre-dawn” or “night”, as the nadir is usually around 4 AM. Terms vary.

* * *

Looking at it from that perspective, it makes sense that there would be something called “inverted typhoid”. This term means a pattern in which the fever is highest in the morning, and reaches a low point in the evening.

Here’s some examples.

From John McCaw’s 1914 Diseases of Children:

During the Second Week: The spleen is enlarged and tender, the abdomen is uniformly swollen, and gurgling in the right iliac fossa is present. The bowels may be relaxed, but quite as often they are constipated. Headache subsides, and delirium at night takes its place. The expression is dull, the decubitus dorsal, the cheeks are flushed, and the child is indifferent to its surroundings, but not in any apparent suffering. Thirst is considerable, and the skin is dry. The temperature varies from 101.5°F to 105°F, rising towards evening and falling again in the early morning, but throughout the attack the highest temperature may be recorded in the morning (inverted typhoid).

From the 1913 Journal of the Indiana State Medical Association:

Dr. Bruggeman: Had patient with a deep phlegmon of the palmar fascia which was incised and drained. Perfect recovery from hand. Two weeks later dull pain in right hypochondrium. Urine normal. Blood showed slight leukocytosis. Examination negative. Lowest temperature from 101 to 103; no chill. Temperature is of the inverted typhoid type. Widal negative. Leukocytosis diminished.

From the 1884 Dublin Journal of Medical Science (describing a fever that isn’t necessarily infectious at all):

Temperature in Insanity.— Extended contributions to this subject have recently been made by Bechterew (Archiv für Psychiatrie, Bd. XIII.) and Hebold. Bechterew has taken the temperature of the rectum with all the precautions suggested by Liebermeister. He finds that in the first stage of melancholia the temperature usually remains normal, or may even rise above it. It has been observed as high as 104°F. By melancholia Bechterew evidently means all cases with delusions of persecution and depression. …In the convalescent period the temperature is usually normal. Sometimes the temperature is extremely variable at the outset of this period, and this usually denotes a sudden improvement in the patient’s condition. In the excited or stuporose period an inverted typhoid fever curve is often noticeable.

In 1867’s Notes on Asiatic Cholera, John Charles Peters viewed “inverted cholera” yet another way.

Some physicians believe that cholera is in some strange way mixed up with intermittent and remittent fevers in India in the East; and with typhoid fever in Europe and the West. Others have even gone so far as to describe cholera as an inverted typhoid fever; it commences with profuse discharges, and the latter is apt to end with them; the one has collapse before the fever, and the other afterwards, &c.; the causes of both are said to be similar with the difference of climate only.

Coincidentally, the first convenient clinical thermometer was manufactured in 1867! So Dr. Peters was writing this in a world before typhoid was known to have an easily-measured daily fever cycle. Even then, it was a disease with a standard pattern of symptoms to which other diseases could be compared and contrasted.

* * *

Okay. “Inverted typhoid” is a symptom, that is not necessarily indicative of typhoid, or even of infection. It means high fever in the morning, and not so high in the evening.

But when you look into “inverted typhoid”, the most significant article on the subject is a piece in the 1898 Medical Record, by Max Goltman of the Shelby County Poor and Insane Asylum, Memphis, Tennessee. For more about the Glasgow-born Dr. Goltman, see the Jewish Historical Society of Memphis and the Mid-Southmax-goltman-md.

Dr. Goltman’s definition of “inverted typhoid” is different.

On September 25, 1897, I was called to see Arthur Z—–, thirteen years of age, white. He was born and brought up in Memphis, and was bright, exceedingly energetic, and of a very nervous temperament. …

He had been indisposed, more or less, for about two weeks prior to my first visit, complaining of throbbing headaches, which were worse in the afternoon and after exercise; and there were loss of appetite, lassitude, drowsiness, disturbed sleep, constipation, and feverishness. Being an ardent cyclist, he made frequent trips far into the country in the hot sun. He recollected having partaken, on several such occasions, of both milk and water at a roadhouse where there had been sickness in the family for some time.

I diagnosed malaria, and prescribed a saline purge and quinine in three-grain capsules every four hours, an ice cap to the head, and a cool bath. About six hours later I received word that the patient was resting nicely and was nearly, but not quite, free from fever. On September 26th he had another chill, which was of a much milder character than that of the preceding day. The quinine was continued until twenty-four grains were taken. This kept the patient comfortable, but with a temperature ranging from 99° to 101°F until September 29th, when he had another violent chill, followed in a short time by fever of 104.8°F. On being informed of this, I made an examination of the blood for malaria.

What seemed like a typical case of malaria is not following the normal pattern, so he looks to see if it really is malaria. The parasites are clearly there in the blood cells. But the symptoms keep changing. I’m no doctor, let alone a 19th-century doctor, so I don’t know why he suspected that malaria was “not accounting for the symptoms present”. Maybe the patient was no longer having chills. Anyway, three days after seeing malaria under the microscope, he looked for other pathogens, and the Widal reaction showed clear signs of typhoid.

But the fever didn’t follow the typhoid pattern any more than it followed the malaria pattern. Here’s Goltman’s temperature chart of “inverted typhoid”.

goltman-inverted-typhoid

As you can see, Arthur Z—- starts out with a fever. But the “Rose Spots” noted on Day 6, normally found in the plateau stage of typhoid fever, are here accompanied by a decrease in fever, quickly reaching sub-normal temperature. At this point Arthur had probably been switched from quinine to heavy doses of cathartics, with the goal of maximizing bowel movements to get rid of the bacteria.

I [have] discussed the eliminative treatment of typhoid fever very extensively, and urged the adoption of the saline cathartics for the purpose of elimination in preference to anything else. I have no reason to change the opinion then expressed. They are undoubtedly best and safest. The patient may get tired of salts; then, for a day or two, calomel may be substituted in broken doses until the bowels move. A good mixture to employ is a drachm each of Epsom salts, Rochelle salts, and compound licorice syrup, repeated if necessary. Four or five movements a day are desirable. This is my main treatment; everything else is subservient to it.

And indeed, as the patient “got tired of salts”, he replaced the cathartic salts with one dose of strychnine, followed by calomel. This coincides exactly with the body temperature returning to normal. Whether the treatment helped or hurt, who knows. The point is that we have, as Dr. Goltman says in his somewhat flowery conclusion, “an Æsculapian paradox — a fever without fever”. In other words, a case of inverted typhoid.

One more quote.

Gentlemen, we have here for analysis a case of atypical typhoid fever ushered in by the clear-cut paroxysms of a malarial infection, which was demonstrated beyond the shadow of a doubt by microscopic examination of the stained and fresh blood of the patient. More typical organisms I have never seen … Having made such a diagnosis in this case, it was somewhat humiliating to be compelled apparently to recede from my position, and inform the anxious family that they had now to nurse a case of typhoid fever. Dr. Pheemster has, however, told me of a similar instance occurring at St. Joseph’s Hospital, and the literature of the subject teems with similar cases, which, unlike my own, however, are somewhat lacking in scientific data and therefore of doubtful value.

First of all, I highly doubt there was ever a person named “Dr. Pheemster”. That sounds like a Robert Benchley character. Second of all, is it necessary to claim that yours is the only reliable case report ever published? It’s not like you have lots of data either. Just one temperature chart.

Finally, another frequently re-printed study, originally in JAMA, mentioned “inverted typhoid” in passing. This was by Dr. George Boody, recounting a typhoid outbreak at the Iowa State Hospital for the Insane (now the Independence Mental Health Institute). Dr. Boody includes a multi-week fever curve similar to that of Dr. Goltman, indicating that to him the term refers to a case of typhoid that progresses from fever to sub-normal temperature. He doesn’t mention malaria or any other comorbidities, saying simply that “[c]ases of typhoid fever are comparatively quite rare, and the subject is deserving of thorough investigation as often and wherever an epidemic occurs… In these two epidemics it occurred but once in forty-three cases.”

* * *

So, we have “inverted typhoid” meaning a case of typhoid where the fever goes down rather than up, because of malaria. Or it means a case where the fever is high in the morning rather than the evening. Or it means a case that does not involve actual typhoid, just a typhoid-esque fever pattern.

Good thing nobody uses the term anymore! Instead, we use the term “typhus inversus”, which meant the same thing as “inverted typhoid” back in Dr. Goltman’s day. Now “inverted typhoid” has died out, but “typhus inversus” remains.

From Khan Abdul Kalam Azad (2011), Changing trends in pattern of presentation with different types of pyrexia and approach to be made. J Dhaka Med Coll 20(1):1-3 (available here):

A diurnal pattern also known as typhus inversus, is the reverse of normal circadian pattern in which the highest temperature is in the morning. It can be found in miliary TB, hepatic abscesses and endocarditis.

The same definition is in ML Kulkarni (2008), Clinical Methods in Paediatrics: Physical Examination of Children (Jaypee Brothers Medical Publishers, New Delhi), page 61 (partially available here), and in the fourth edition of Moffet’s Pediatric Infectious Diseases: A Problem-oriented Approach (2005) by Fisher, Boyce and Moffet (partially available here).

Calling it “typhus inversus” is less confusing than implying that the person suffering from TB or salmonellosis also has some odd form of typhoid.

But it’s still confusing.

Gnezda, Pittaluga, Zipfel, Goré, and Berschaffelt back me up on this

Sometimes you see an old scientific paper that does a lot of name-dropping. Instead of saying something like “we used an isothermal calorimeter incorporating a constant-flux modification”, it’ll say “we used a Stanpfer device incorporating a Billigs modification, as demonstrated by Reemis”. I guess this sort of thing is still common in chemistry where every reaction is named after some long-dead German or Japanese person, but to me it seems to hearken back to the days when science was a pursuit for punctilious aristocrats and eccentric showmen.

indol-gnezda

W. L. Holman and F. L. Gonzales are classic name-droppers.

Some excerpts from A Test For Indol Based on the Oxalic Acid Reaction of Gnezda (found in the November 1923 Journal of Bacteriology, 8(6):577-583):

Gnezda described a pink or purple color reaction formed by the union of oxalic acid and indol in 1899. It is not clear whether Morelli or Pittaluga first applied this reaction to bacteriologic studies.

Morelli does not refer to Gnezda’s work and Zipfel thought that this was simply a return to the principle of the Crisafulli pine splinter hydrochloric acid procedure.

Verschaffelt used the method to demonstrate indol from jasmine and orange blossoms.

Konrich is reported to have found it unsatisfactory. Zipfel, Baudet and Freund obtained results which compared favorably with other standard methods.

The results given with the Salkowski and Ehrlich tests frequently fail to agree. It would appear from the studies of Frieber and many previous workers that the Salkowski test is not reliable as a test for indol, since it gives a reaction quite similar when indol acetic acid is formed from the tryptophane molecule. The Ehrlich-Böhme test has been found to react with other compounds than indol.

Of course, this paper is under no obligation to explain what the “Salkowski test”, the “Ehrlich test”, or the “Ehrlich-Böhme test” are. The audience was probably familiar with these things. The concept of an indol test, or indole test as we call it now, is not that complicated, and in fact is still performed today, with continued improvements and modifications.

And most of the names dropped here are found in the bibliography. In fact, here’s the bibliography.

indol-bibliography

That’s actually a very long bibliography for a 1923 paper. It would take some effort to find all those papers to see what the authors are talking about, if you’re not actively keeping up with the various permutations of the indole test.

Just as important… most of those papers are not in English. We can’t be sure because there’s no paper titles listed, only the names of the journals. But I count 5 references to English-language journals (J Biol Chem, J Bacteriol, Indian J Med Res); 4 French; 2 Italian; 1 Spanish; 2 Dutch (Folia Microbiol Delft?), and 14 German. There were some synopses of the foreign literature in American journals — unnecessary nowadays when almost everything is in English. But it seems like you had to at least read both English and German (and French?) to follow the scientific literature.

* * *

Personally, I want to know more about “the Crisafulli pine splinter hydrochloric acid procedure”. That sounds great. So evocative of austere old-world elegance. If anyone has a copy of the 1895 Rivista d’Igiene e Sanità Pubblica, containing Gugliermo Crisafulli’s influential article “La reazione rossa del legno di pino per la ricerca dell’indolo nelle culture in brodo dei microrganismi”, please send it in. Or send in a translation.

Data Update: Look at the polio fly

In our last Data Update, the table that I turned into figures was not a bad table. It was pretty clear. It just contained some unnecessary information, and was spread across two pages, which is always bad. Today, the table in question is really hard to interpret. I could not make heads or tails of it without going over the text, piece by piece. It’s from a 1943 paper (1) in the Journal of Infectious Diseases.

* * *

Like the pictures of cockroach feeding contraptions, this data comes from a laboratory that was using what could be called “experimental epidemiology” techniques, to figure out how much of a public health hazard these bugs really are. To see if bugs could actually ingest, preserve, and spread germs.

This time the bug is the common house fly. Thomas Francis Jr. had just joined the faculty of the University of Michigan from NYU, and with technician Robert Rondtorff he conducted this study in the interest of public health. Soon Francis would be supervising graduate student Jonas Salk, with whom he worked a lot in the 1940s and 1950s, as you might be able to guess from the fact that Dr. Francis now has a Wikipedia page.

By 1943 we knew that polio was spread by filth and tainted water (the fecal-oral route, as we call it). But flies feed on that stuff, and fly around. Does the virus replicate inside the flies, like malaria? This paper established that when flies ingest poliovirus, the virus disappears from the digestive system within 2 days. And therefore, flies probably aren’t making the poliomyelitis epidemic worse. The significance of these findings is indicated by the introduction to a 1950 paper (2) by children’s television character “Herbert Hurlbut”.

Poliomyelitis virus has been isolated from filth-frequenting flies caught in nature during several epidemics in recent years.(3-5) When the Lansing strain of the virus was fed to house flies under experimental conditions, virus was not recovered after 48 hours.(1,6) Recently Melnick and Penner (7) fed virus in human stools to the blowfly Phormia regina and were able to recover it from the flies after about two weeks[.]

* * *

First of all, their experimental system. Feed the flies on polio-infected material. To make sure there was a lot of polio in the flies’ diet, they did not use excrement from polio-infected animals. They used “a 10% suspension of infected [mouse] spinal cords in boiled milk”. In the not-entirely-robotic prose of scientific papers of the era, they say they “offered” this to the flies. Earlier they had just diluted the mashed spinal cords in saline solution, and the flies “did not feed readily”, so they switched to a solution in milk, which the flies found far more appealing.

For this experiment, the flies enjoyed their neuronal polio-milk for 1 hour, after which it was removed and they were allowed to feed on regular milk if they wanted to. The scientists then waited either 0, 2, 7, 13, 25, 49, 120, 240, or 480 hours. After each of these periods, a certain number of flies were killed, and their abdomens were cut away from the rest of the body. Earlier in the paper it had been established that poliovirus does not leave the abdomen (gut) of an infected fly and reach the rest of the body (thorax and head). Now they are looking to see how much polio virus is in the infected flies.

To get an extract containing poliovirus, they took the fly parts and ground them in saline solution using a mortar and pestle. For best results, they ground the samples with alundum (an abrasive preparation of aluminum oxide). They added some ether (diethyl ether, I assume) to the solution, and let it sit in the refrigerator until it was “bacteriologically sterile”. I’m not familiar with the use of ether to remove bacteria, but it looks very suitable to this procedure.

Experimentally and clinically, ether, whether in its vaporous or liquid state, has been proved to have a bactericidal action. Spore bearing organisms, however, are strongly resistant to it. (7)

Not only did ether treatment remove bacteria from the extract, but it also removed most other viruses. Poliovirus, as a non-enveloped virus, is resistant to ether, a substance which destroys the membranes of enveloped viruses. Here’s a table that summarizes, as of 1949 (8), which viruses are ether-resistant.

andrewes-horstmann-ether

from Andrewes and Horstmann (1949)

Note that most viruses are ether-sensitive and should be removed by this sort of treatment, meaning you’re left with polio, papilloma, bacteriophages, and a few other viruses. Including some, but not all poxviruses, for reasons that are unclear to me. Parapoxviruses (myxoma, BPS virus) are ether-sensitive, and orthopoxviruses (smallpox, vaccinia) are ether-resistant, which is still the dogma (see Chapter 21 of Medical Virology). And chordipoxviruses are maybe one or maybe the other. The above table puts sheep-pox and goat-pox (chordipoxviruses) in the resistant group, whereas Plowright and Ferris (9) say that sheep-pox (SP) and lumpy skin disease (LSD) viruses are ether-sensitive.

from Plowright and Ferris (1959)

from Plowright and Ferris (1959)

Anyway, poliovirus is definitely ether-resistant. So this extraction method is useful for studying this particular virus [here’s another example (10)].

It wasn’t possible in 1943 to measure the amount of virus by using a plaque assay, as we do now (applying a solution of virus to a plate of cells and seeing how many plaques [empty spots] form in the cells by being killed by virus). What the authors do instead is infect mice with the fly extract and see if they become paralyzed and/or dead. In Table 1 they established that the virus survives in the flies, but only in the abdomens. Here’s Table 2.

rondtorff-table-2

So one half of the table is data from the flies, and one half is data from the mice, right? That’s what I thought. But no. This table is made up of almost entirely unnecessary information, and only makes one point. I’ve taken the liberty of highlighting the important parts.

rondtorff-table-2-2

In the text, Rendtorff and Francis go into great detail about how they prepared the ground fly mixtures so they could be compared fairly. They weighed the samples before grinding, and added more or less saline depending on how many fly abdomens were in the sample, and what their weight was. In deciding how much saline to add to dilute the sample, they also factored in what the average unfed weight of an abdomen should be, which should be the same for all the flies. This process was to normalize the data, which could be thrown off by the flies’ unequal eating and excreting habits. I feel like pooling together somewhere between 44 and 66 fly abdomens would already take care of the issue of some flies eating more than others, but they did this statistical technique to account for possible variation.

Most of this table is the raw data they used to do the normalization. But… I don’t need to know what the weight of the fly abdomens were. Or how much saline was added. And I definitely don’t need the “Calculated Unfed Weight of Abdomens” column, which is nothing but the “No. of Flies Sampled” column multiplied by the average weight of an unfed abdomen. It’s good to present raw data, I guess. But this data is not important. Maybe it should be separated from the important data.

The least important data of all is the dates on which the experiments were performed. This is something that absolutely never shows up in research articles anymore. In fact, we may have gone too far in the opposite direction, pretending that we did experiments in a certain order because it makes for a better narrative.

All that matters here is two independent variables and one dependent variable. The independent variables are:

  • Temperature of the incubators in which the flies were living, eating and loving life. (25, 30, or 35 °C)
  • Number of hours the flies were allowed to live, between eating the polio-infected meal and being killed. (0, 2, 7, 13, 19, 25, or 49 hours)

The dependent variable is:

  • Time of death (or paralysis) for a mouse injected with fly extract

So all we really need is a bunch of survival curves.

polio-survival

Figure 2: Poliomyelitis virus is destroyed by the fly digestive tract within 2 days, irrespective of temperature. Flies were fed on a suspension of infected spinal cord in milk for 1 hour. Flies were then incubated in a chamber at 25, 30, or 35 degrees Celsius, for 0 (positive control), 2, 7, 13, 19, 25, or 49 hours. At each timepoint, between 45 and 65 flies in each chamber were killed, and abdomen tissue was extracted using ether. After dilution in saline and evaporation of the ether, each sample was used for intracerebral infection of 9 or 10 mice. Twice a day, mice were monitored for death or paralysis.

* * *

1. Rendtorff RC, Francis T Jr (1943). Survival of the Lansing strain of poliomyelitis virus in the Common house fly, Musca domestica L. J Infect Dis 73(3):198-205.

2. Hurlbut HS (1950). The recovery of poliomyelitis virus after parenteral introduction into cockroaches and houseflies. J Infect Dis 86(1):103-104.

3. Trask JD, Paul JR (1943). The detection of poliomyelitis virus in flies collected during the epidemics of poliomyelitis. J Exp Med 77:531-544.

4. Sabin AB, Ward R (1941). Flies as carriers of poliomyelitis in urban epidemics. Science 94:590-591.

5. Melnick JL (1949). Isolation of poliomyelitis virus from single species of flies collected during an urban epidemic. Am J Hyg 49:8-16.

6. Bang FB, Glaser RW (1943). The persistence of poliomyelitis virus in flies. Am J Hyg 37:320-323.

7. Saliba J (1918). Ether therapy in surgical infections and its effect on immunity. New York Med J 107:157-160.

8. Andrewes CH, Horstmann DM (1949). The susceptibility of viruses to ethyl ether. Microbiology 3(2):290-297.

9. Plowright W, Ferris RD (1959). Ether sensitivity of some mammalian poxviruses. Virology 7(3):357-358.

10. Ward R, LoGrippo GA, Graef I, Earle DP Jr (1954). Quantitative studies on excretion of poliomyelitis virus: A comparison of virus concentration in the stools of paralytic and non-paralytic patients. J Clin Invest 33(3):354-357.

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”.

* * *

bass-watkins-test

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.

bass-watkins-figure1

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.

von-pirquet-reaction-1914

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.

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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.

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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.