Amboceptor

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

Tag: JAMA

Yellow fever stops at the Miami airport.

staining-mosquitoes-before-liberating

Initially, new infectious diseases could spread only as fast and far as people could walk. Then as fast and far as horses could gallop and ships could sail. With the advent of truly global travel, the last five centuries have seen more new diseases than ever before become potential pandemics. The current reach, volume and speed of travel are unprecedented, so that human mobility has increased in high-income countries by over 1000-fold since 1800. Aviation, in particular, has expanded rapidly as the world economy has grown, though worries about its potential for spreading disease began with the advent of commercial aviation. [1]

Podcasts are great. But in the world of science podcasts, many are simply boring because there is only one person talking, or one person interviewing another person. Unless it’s slickly produced and edited (the Nature or Science Times podcasts), I’ll quickly lose interest.

It’s better when the podcast is three or four people who know each other, having a conversation. This is is the format of the TWi[X] series, particularly the flagship This Week in Virology. TWiV co-host Dickson Despommier, though not a virologist, contributes a big-picture point of view when the show leaves its territory of basic lab science and moves into epidemiology and patterns of disease outbreaks. Here’s his lecture on how West Nile arrived in North America and spread from state to state, energized by a very hot summer at the Bronx Zoo.

More than once I recall Dr. Despommier pointing out that though it’s all well and good to model how a epidemic might spread by looking at the day-to-day movements of people and mosquitoes, the most powerful and mobile disease vector is… the airplane.

That fact has become ever more clear with the SARS and West Nile outbreaks, as we used genetic analysis to track them across the continents in real time. But you might be surprised at how long ago people recognized the airplane as a challenge to disease control.

* * *

80 years ago, yellow fever was the archetypal mosquito-transmitted disease. Particularly as the disease was endemic in some parts of the world (e.g. South America), but not in others (e.g. North America), despite both places being home to the vector, Aedes genus mosquitoes. A 1934 article [2] coauthored by Henry Hanson, veteran of battles against yellow fever on three continents, points out that dengue fever (also transmitted by Aedes) had reappeared in Florida after an eleven-year absence, and yellow fever could do the same at any time.

dengue-1922-1934-tampa-miami

Hanson and coauthor T. H. D. Griffitts go on to chide the cities of Florida and Georgia for their complacency in providing countless unsupervised water vessels in which the mosquitoes can reproduce.

Practically every urban community in the South has its array of artificial containers, from flower vases to catch basins, cuspidors and discarded automobile tires, producing Aedes aegypti. For example, the city of Tampa in eight weeks reported finding 1,091,823 containers (potential mosquito “hatcheries”). Of these, larvae were found in 20,864, or approximately 2 per cent. This was an unusually dry season (average rainfall of 1 1/2 inches a month for this period). It is interesting to note that in Miami for a like period only 56,598 potential breeding containers were reported, with 38,401, or 68 per cent, of the total actually breeding.

So I made an error above. The vessels aren’t quite “countless”. There are 1,091,823 of them in Tampa, give or take a dozen.

Though concerned that yellow fever could reach North America (again), Hanson and Griffitts are more concerned about India. The disease is only known in jungle areas of Brazil, Colombia and Bolivia (they claim); by comparison, Africa is its ancestral “home” and India, fairly nearby by plane, is virgin territory for yellow fever epidemics.

Today there is a feeling of concern … that Old World territory, where the vectors abound and where yellow fever has never before stalked, may experience widespread and devastating epidemics. One infective mosquito traveling in an airplane from the home of yellow fever (Africa) to India could be the spark to start the conflagration.

Thus there was considerable surveillance for mosquitoes and infected passengers at airports worldwide.

The earliest article I can find specifically discussing airplanes as disease vectors is from 1930, the first [3] of two identically titled 1930s editorials in the American Journal of Public Health. I don’t know if the international rules discussed here were enacted, but they show awareness that quarantine and disinsectization measures developed for ocean travel need to be multiplied and intensified to cope with passenger aircraft. Click for a bigger view.

airplane-and-yellow-fever-1930

* * *

For the purpose of determining whether or not mosquitoes are carried in airplanes, and, if so, to what extent, the distance of such transportation, the species of mosquitoes, and the type of planes on which they are carried, the United States Public Health Service began, on July 23, 1931, the inspection of all airplanes from tropical ports arriving at the airports of the Pan American Airways System at Miami. [4]

An anecdote from less than a year later illustrates how the search for mosquitoes had become a normal part of a plane’s arrival in the U.S. from South America.

A very Normal Rockwell scene of mosquito inspection. From LIFE Magazine, 5/27/40.

A very Normal Rockwell scene of mosquito inspection. From LIFE Magazine, 5/27/40.

The story of “the first international aerial hitchhiker” is as follows.

Paul Kaiser, 25, tried to immigrate from Czechoslovakia to America via a circuitous route. First, he got to the German port of Bremen, from which he sailed to Colón, Panama on an ocean liner. Sneaking into the nearby Canal Zone airport, he climbed into the baggage compartment of a “big 22-passenger Commodore plane”. The plane first landed in Barranquilla, Colombia, where he was not detected. It then landed in Kingston, Jamaica, where he was not detected. Finally, after Kaiser had spent 2 days without food, he landed in Miami. However, in Miami “a very thorough inspection is given every plane for mosquitoes, for there is always danger of the deadly yellow fever mosquito surviving the short trip and landing in the United States, a most undesirable immigrant”. Check out the April 1933 issue of Flying magazine for more details.

A few months earlier, T.H.D. Griffitts (coauthor of the aforementioned Henry Hanson article) performed the first experiments on mosquito transport by aeroplane. These were published in late 1931, in an enjoyably conversational article in Public Health Reports [4].

Dr. Griffitts, you don't really have to describe the experiments you WANTED to do but then decided were unnecessary.

Dr. Griffitts, you don’t really have to describe the experiments you WANTED to do but then decided were unnecessary.

T.H.D. Griffitts (and J.J. Griffitts — his son?) start out by describing all the mosquitoes observed on normal commercial flights between July and September of 1931. Most were Culex quinquefasciatus, but several other species were observed including Aedes aegypti. In fact, a later Griffitts paper [5] indicates that the historic first mosquito discovered on a Miami-bound plane was Aedes aegypti. (It’s somewhat confusing that the insect apparently arrived on a “ship from San Salvador, El Salvador”, but I think that at this time the word “ship” could be used for aircraft. And San Salvador is definitely not a port city.)

Griffitts also put mosquitoes on three planes departing from San Juan, Puerto Rico, and looked for them upon arrival in Miami. Today it takes 2.5 hours to fly from SJU to MIA, but in 1931 they stopped at three other airports along the way and the average travel time was over 10 hours. A total of 100 mosquitoes were labeled with eosin dye (to distinguish them from non-experimental and Miami-resident mosquitoes). 22 were observed upon arrival in Miami, after 1,250 miles of flying, opening of doors and hatches, loading and unloading of luggage, etc. It seems obvious that the insects can be imported… but these experiments prove it.

* * *

Eight years after 1930’s “The Airplane and Yellow Fever” editorial, the American Journal of Public Health published another one [5], describing the policies for infectious mosquito control in Khartoum, Miami, and “the French Colonies and Mandates”.

As the 1930s progressed and India remained devoid of yellow fever, public health doctors kept amplifying their alarms about how devastating such an epidemic would be. From the 1938 editorial:

greatest-menace-in-the-world-today

75 years after that was published, yellow fever still hasn’t swept through India.

In 1938, the number of people who’d been vaccinated against yellow fever was less than a hundred thousand, mostly in Brazil. It had taken a long time to make a vaccine strain of YFV that was weak enough that it could be given as a live vaccine, but the 1938 trial (run by the Rockefeller Foundation) was successful, and in 1942 the number of people vaccinated was over 10 million. For his two decades of work on the vaccine, which along the way required multiple basic science breakthroughs for it to be manufactured in large quantities, Max Theiler of Rockefeller University received the 1951 Nobel Prize in Physiology or Medicine. Read more here [6] about Theiler’s story; read more here [7] about the many problems and hurdles that were overcome to end up with a safe vaccine. (I think the second one is open-access and the first one isn’t.)

Within a decade, though complications from the vaccine were common and the virus stayed endemic in the tropics, Theiler’s YFV vaccine had eliminated yellow fever as a source of large-scale epidemics.

* * *

Coda: As early as the airplane was recognized as a vector for disease… it was harnessed as a weapon against disease.

From a 1932 report in Science (8), Joseph Ginsburg of the New Jersey Agricultural Experiment Station explains how large regions of standing water that previously were inaccessible to mosquito control workers can now be reached by plane, so that the surface can be coated with larvicidal pyrethrum or oil.

cheesequake-killing-film

 

* * *

1. Tatem AJ, Rogers DJ, Hay SI (2006). Global transport networks and infectious disease spread. Adv Parasitol 62:293-343.

2. Griffitts THD, Hanson H (1934). Significance of an epidemic of dengue. JAMA 107(14):1107-1110.

3. Editorial (1930): The airplane and yellow fever. Am J Public Health 20(11):1221-1222.

4. Griffitts THD, Griffitts JJ (1931). Mosquitoes transported by airplanes: Staining method used in determining their importation. Public Health Rep0rts 46(47):2775-2782.

5. Editorial (1938): The airplane and yellow fever. Am J Public Health 28(9):1116-1118.

6. Norrby E (2007). Yellow fever and Max Theiler: The only Nobel Prize for a virus vaccine. J Exp Med 204(12):2779-2784.

7. Frierson JG (2010). The yellow fever vaccine: A history. Yale J Biol Med 83(2): 77-85.

8. Ginsberg JM (1932). Airplane oiling to control mosquitoes. Science 75(1951):542.

 

Advertisements

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.

* * *

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.

Data Update: Typhoid ice cream

To this day, the safety of the ice cream supply is a field of inquiry among microbiologists, as can be seen in recent findings from Croatia (1), Zimbabwe (2) Argentina (3), and other places (4,5). Here in the U.S. there’s not as much concern; as far as I can tell the last outbreak of food poisoning caused by mass-produced ice cream in a rich Western country was in 1994 (6), when beloved Minnesota company Schwan’s Ice Cream was transporting ingredients in unsterilized egg tankers. Since then there have been outbreaks on farms, such as two big ones in Belgium and Wales. Most are like the Belgian case (7), which was blamed on ice cream produced right there on the farm, rather than industrially. The Welsh case (8) was blamed not so much on the ice cream itself, but on ice cream’s notorious ability to make children’s hands sticky, turning them into repositories for farm filth. A comparison between desserts in Houston and Guadalajara (9) suggests that the US is fortunate not to have this problem. But back in the 1910s and 1920s it was a different story.

Looking at old tables of contents from the Journal of Bacteriology, it’s striking how many articles are about milk. Even more than other food products, there was a need for science to improve the safety of milk, cream, cheese, etc. Pasteurization was established as useful in the late 19th century, but it would not become widespread until a 1913 typhoid epidemic in New York. This convinced local authorities that the improvement in public health outweighed people’s concerns about what boiling milk did to its taste and possibly its nutritional content (for more on this see Neatorama, “The Fight for Safe Milk”). But still, outside major urban centers where access to fresh milk was limited, people figured that raw milk was fine because the farms were nearby.

And ice cream was contaminated, too. By tuberculosis, typhoid, Bacillus coli, and others. But how big a problem was it, really? The bacteria wouldn’t multiply if they were frozen. But would they actually be killed?

The results on this were basically in by 1926, when Michigan State professor Frederick Fabian collected them in a great review in the American Journal of Public Health (10). Some excerpts:

The rapid rise of the ice cream industry and the general use of ice cream as a food in the past few years has added another item to the public health official’s responsibilities. If an epidemiologist had been tracing an epidemic a few years back, he could have practically left ice cream out of consideration. However, today it has become such a common article of diet that it should be taken into account in any work of this nature.

…Although there has been a vast amount of work done to show that pathogenic bacteria are not readily killed by freezing, yet, due to the nature of the product ice cream has not always been considered a serious source of bacterial infection. This view is held especially among laymen and those not familiar with the facts.

…To test out the extreme temperature which pathogenic bacteria could withstand Macfayden and Rowland (11) subjected the same organisms to the temperature of liquid hydrogen (-252° C.) for 10 hours without any appreciable effect on them. A great deal of work has been done also to show that pathogenic bacteria live for a considerable length of time in ice and a great many epidemics have been traced to this source.

…In practically every city of any size today pasteurization of the milk supply is required by ordinance. The time and temperature are also pretty well established. There are not many such ordinances or laws pertaining to ice cream in most cities or states. …Many of the old experienced producers from their experience with milk have the good judgment to pasteurize the ice cream mix. However, the unscrupulous and the ignorant are allowed to do as they please.

In short, by 1926 public health authorities have got a handle on milk contamination, but for the very reason that ice cream seems safer (nothing can grow in it! most of the time), it has not been regulated and may now be more of a risk than milk.

Along the way, Fabian lists all known ice cream-associated outbreaks, and describes the experimental evidence for bacterial survival in frozen ice- and cream-like substances. Let’s clarify those results, now that we have the ability to make graphs.

* * *

Four experimental papers are mentioned. Mitchell 1915 (12), Bolten 1918 (13), and Prucha and Bannon 1926 (14) use Salmonella Typhi (or “Bacillus typhosus“, or “Bacterium typhosum“). Davis 1914 (15) uses hemolytic Streptococcus. I found two later papers in the Journal of Dairy Science by G. I. Wallace of the University of Illinois, but as he himself seems to think his results are unimportant, we won’t bother with them.

G. I. Wallace (1938) is not exactly making a big effort to inflate the importance of his results here, is he?

G. I. Wallace (1938) is not trying very hard to inflate the importance of his results.

O. W. H. Mitchell (1915) doesn’t even have a table in his paper. He describes six experiments which involved similar ice cream preparation and storage, but with different ingredients. For each experiment, he introduced some typhoid bacilli to the ice cream mixture, checked after 24 hours of freezing to see how much bacteria there was, and continued measuring until “the last positive examination” for typhoid bacilli. This seems like a badly-controlled series of experiments, since the amount of bacteria introduced into the cultures varies widely between experiments, and I can’t tell the difference between Experiment 1 and Experiment 2. Apparently in Experiment 2, “Ice cream made with 1 pint of thin cream, one-half cup of sugar and 1 tablespoonful of vanilla was treated similarly to the ice cream in Experiment 1.” But… all three of those ingredients are also in Experiment 1. Because of the order things are listed in, my guess is that Experiment 2 has an extra 1/2 cup of sugar.

Also the description of “Flake” powder does not enable colleagues to easily replicate the experiment.

Flake is a powder prepared by the Murray Company, 224 State Street, Boston. According to a circular accompanying the powder, the preparation “is a pure, wholesome powder, which can always be relied on, and is essential in making an exquisitely smooth ice cream.”

That doesn’t help. Anyway, the jumble of descriptive paragraphs can be entirely summed up in this table.

ice-cream-mitchell-1915-table

O. W. H. Mitchell’s 1915 data, presented in no particular order

Guess what! That tells us nothing. Pasteurization didn’t help; adding gelatin made things worse somehow; and extra sugar led to less bacteria at the late timepoint. Also, there’s no more bacteria after a couple weeks than there was at 24 hours, even though “After a few days [the samples] began to lose their sweetness of odor, and at the time of the last examinations they gave off mildly unpleasant odors.”

Yes, this was while frozen. They were only slightly below freezing (-3° to -4° Centigrade). Sample size is 1. Total waste of time, I say with 98 years of hindsight.

* * *

Let’s move on to Bolten (1918). He looks at both typhoid and diphtheria bacilli. He shows even less data than Mitchell, but at least the experiments make sense. Basically, small containers of frozen cream (not ice cream, I guess there was no sugar or vanilla) had been inoculated with a growing culture of typhoid, at a 10:1 ratio of cream to bacterial broth, and “immediately placed in a brine tank”. They were “partly melted” daily, and a sample was taken to see how many typhoid bacteria were growing. You’d think that one of the advantages of ice cream as an experimental system is that you don’t have to thaw it in order to take a sample. But that was their procedure.

According to more than one article in the Jan-June 1904 issue of Ice and Refrigeration Illustrated (mental note: look for blog topics in Ice and Refrigeration Illustrated), a “brine tank” typically froze things to between 8 and 16 degrees Fahrenheit (-13° to -9°C). So, quite a bit colder than the ice cream in Mitchell’s study. This type of freezing is pretty good at killing bacteria, given that they started with a substance that was fully 10% bacterial broth. After 2 weeks they had a 50% reduction in colonies; after 4 weeks they had a 95% reduction; and after 10 weeks two of the four containers had no detectable germs at all. Maybe it’s not so much the low temperature as the daily freeze-thaw cycle killing the bacteria.

Skipping gracefully over the diphtheria portion of the paper (which is more confusing), our last entry on ice cream typhoid is the most data-intensive, by Prucha and Brannon of the University of Illinois in the relatively rigorous Journal of Bacteriology. (The other two are in medical journals… who cares about lab experiments in those?) They standardize their experiments by mixing bacteria with ice cream mix, incubating, and freezing it when it gets to 25 million bacteria per cubic centimeter. They keep it in a “hardening room” which fluctuates between -8° and 8°F (-22° to -13°C) – the coldest freezing conditions we’ve seen so far. They take samples not every day, but at increasingly sparse intervals – and they actually show us their data in a table. Which can easily be turned into a graph.

prucha-brannon-table

I should use the word “germs” more often in writing about these things. I forget about the word “germs”.

A summary:

prucha-brannon-graph

Here we see something much closer to Bolten’s “bacteria are mostly killed by freezing”, rather than Mitchell’s “bacteria keep growing in the cold”. And this wasn’t with constant refreezing and rethawing, either. Just low temperatures.

You may notice that the number increases 3-fold between day 134 and 165. Well, they explain that too.

It will be observed in table 1 that the samples taken when the ice cream had been in storage for 165 days gave higher counts than the previous samples. To check this point, another set of samples was taken five days later which again gave similar counts. An inquiry brought out the fact that one of the attendants had removed the experimental ice-cream a few days before to an adjoining room for an hour and one-half. This room had a temperature of 40°F. The ice cream did not melt. Whether there was any multiplication of the germs at this time could be determined.

The conversation that led to that passage:

Prucha: What happened here?

Brannon: I looked into that. You won’t believe it. One of the fellows in the dairy husbandry program, who we told to keep an eye on the freezer…

Prucha: Yes?

Brannon: Well, some of his fraternity brothers let him know it would be a great joke to take the experimental ice-cream into the common room and start eating it.

Prucha: Oh, goodness. Doesn’t he know we put typhoid in it?

Brannon: I think we were a little too excited when we found out freezing had reduced the germs by 99%.

Prucha: So did it melt? Is that why the numbers are high?

Brannon: Luther says he told the boob to put it back in the freezer before it got soft. It was out for maybe 90 minutes.

Prucha: Undergraduates! Damned impudent wastrels!

Brannon: Do we really need “attendants” for this experiment at all?

* * *

1. Mulić R et al (2004). Some epidemiological characteristics of foodborne intoxications in Croatia during the 1992-2001 period. Acta Med Croatica 58:421-427.

2. Igumbor EO et al (2000). Bacteriological examination of milk and milk products sold in Harare. Afr J Health Sci 7:126-131.

3. Di Pietro S et al (2004). Surveillance of foodborne diseases in the province of Rio Negro, Argentina, 1993-2001. Medicina [B Aires] 64:120-124.

4. Gücükoğlu A et al (2012). Detection of enterotoxigenic Staphylococcus aureus in raw milk and dairy products by multiplex PCR.
J Food Sci 77:M620-623.

5. el-Sherbini M et al (1999). Isolation of Yersinia enterocolitica from cases of acute appendicitis and ice-cream. East Mediterr Health J 5:130-135.

6. Hennessy TW et al (1996). A national outbreak of Salmonella enteritidis infections from ice cream. N Engl J Med 334:1281-1286.

7. De Schrijver K et al (2008). Outbreak of verocytotoxin-producing E. coli O145 and O26 infections associated with the consumption of ice cream produced at a farm, Belgium, 2007. Euro Surveill 13:8041.

8. Payne CJ et al (2003). Vero cytotoxin-producing Escherichia coli O157 gastroenteritis in farm visitors, North Wales. Emerg Infect Dis 9:526-530.

9. Virgil KJ et al (2009). Coliform and Escherichia coli contamination of desserts served in public restaurants from Guadalajara, Mexico, and Houston, Texas. Am J Trop Med Hyg 80:606-608.

10. Fabian FW (1926). Ice cream as a cause of epidemics. Am J Public Health (N Y) 16:873-879.

11. Macfayden A & Rowland S (1900). A further note on the influence of the temperature of liquid hydrogen on bacteria. Lancet 156:254-255.

12. Mitchell OWH (1915). Viability of Bacillus typhosus in ice cream. JAMA LXV:1795-1797.

13. Bolton J (1918). Effect of freezing on the organisms of typhoid fever and diphtheria. Pub Health Rep 33:163-166.

14. Prucha MJ, Brannon JM (1926). Viability of Bacterium typhosum in ice cream. J Bacteriol 11:27-29.

15. Davis DJ (1914). The growth and viability of streptococci of bovine and human origin in milk and milk products. J Inf Dis 15:378-388.