Amboceptor

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

Category: Experimental

Data update: How dyestuffs make stuff die

churchman-parafuchsin

This year, antibiotic researcher Mark Wainwright published a “Discussion” in the International Journal of Antimicrobial Agents, suggesting that we should take another look at a subject that he works on but most scientists lost interest in 6 decades ago: highly toxic synthetic molecules that can be used as local antiseptics and have distinctive bright coloration. What he calls “photoantimicrobials” represent a subset (those that can be targeted more precisely, because they are activated by light) of therapeutic molecules that earlier generations simply called flavines, anilins, or “dyes”.

Wainwright tells us:

Browning introduced the application of the basic dyes named above [acriflavine, crystal violet and brilliant green] to battlefield wounds in 1914/15. The aim was always to achieve antisepsis and healthy wound closure, and the approach was also used in pre-operative sterilisation. The dyes used thus represent local antiseptics. Importantly, however, they were an improvement on the available hypochlorite solutions owing to the fact that they were not inactivated by fluids associated with wounds, such as serum. Fleming’s rebuttal of this work, noted above, was based purely on laboratory measurement. However, the disinfection of real wounds and the concomitant recovery of patients could hardly be argued against.

By far the majority of clinical work involving dyes was carried out before 1945. At this juncture, the penicillins and other antibiotics or natural product-derived antibacterial agents became the driving force in infectious diseases therapy. From the distance of the early 21st century, it is apparent that these wonder drugs have not been used correctly and that their overuse has allowed a much more rapid development of resistance mechanisms than might otherwise have been the case.

In terms of modern healthcare, dye therapy might be considered obsolete and ineffective, and this would indeed be the case if the performance criteria were still the same as in Browning’s day. However, recent research has developed a different approach, one that uses locally applied dyes in conjunction with light to provide a microbial killing effect. Such dyes may be referred to as photodyes or, more properly, photosensitisers. Given their remarkable utility in the field under discussion, they are commonly called photoantimicrobials.

The photoantimicrobial effect has been known in the laboratory since the turn of the last century, but has only been investigated realistically since the early 1990s and is currently proposed foruse in oral and ENT (ear, nose and throat) disinfection. Its remarkable activity lies in the in situ production of reactive oxygen species (ROS) on illumination. The highly reactive nature of species such as singlet oxygen, the hydroxyl radical and the superoxide anion ensures rapid oxidative damage to simple cells, sufficient to guarantee cell death. More importantly, there are no known microbial resistance mechanisms able to combat ROS produced in this way.

Furthermore, among the lead compounds used in photo antimicrobial discovery are the same dyes used by Ehrlich and Browning; methylene blue, crystal violet and acriflavine.

Brilliantly colored solutions for treating localized infections and washing out wounds may be making a comeback. And not just to treat pets, for which acriflavine, for example, is now used. In preparation for that retro craze, let’s look at some of the original pre-Depression research. churchman-gazette

Most of it is in German, which I will ignore.

Much of the non-German work on “dye therapy” was led by urologic surgeon John W. Churchman of Yale University. At the top of this post is a rare color photo of bacteria in a Petri dish from the time when Julius Richard Petri (1852-1921) was still alive. This comes from a 1913 Journal of Experimental Medicine paper by Churchman, The Selective Bactericidal Actions of Stains Closely Allied to Gentian Violet, whose figures are otherwise black & white. Even the limitless moneybags of Old Elihu and the Rockefeller Institute only extended to printing one color figure.

* * *

Churchman’s papers are not very quantitative, but other “dye therapy” researchers did large-scale comparisons of multiple colored antibiotics, at multiple concentrations, against multiple bacterial species.

krumwiede-title

On January 1, 1914, the Journal of Experimental Medicine published the Observations of Josephine S. Pratt and Charles Krumwiede Jr. (not to be confused with their Further Observations, published 4 months later). They established a method for testing large numbers of drug/bacteria combinations while minimizing the amount of agar needed, by putting two samples in each Petri dish, slanted away from each other. Here they describe the process in vague terms that would be better presented as a 5-minute video.

As a routine a batch of agar was selected which was found especially suitable for the more feebly growing [bacteria]. To the hot agar an appropriate volume of a watery solution of dye was added to give the final dilution desired. The same agar was used in each experiment.

The most convenient and economical method was found to be as follows. One unopened Petri dish was used to tilt up one side of a second dish. In the opposite side was poured just sufficient agar to give a satisfactory slant. This dish was covered and used to tilt up a third dish, and so on in a row. After the agar had set, slants were poured in the other side of the Petri dishes which were tilted in the reverse direction. In this way two mixtures of agar could be used in the same dish, very little agar being required for each slant.

After inoculating all these plates with bacteria, they used the eye test. Compared to a control plate with no dyestuffs, how much did the dye prevent the bacteria from growing after 18 to 24 hours? Not at all? Was it “restrained”? “Markedly restrained”? Completely eliminated?

Here’s Table 1.

dyestuffs-original-table-1

That is a lot of information. 11 drugs, tested against 30 bacteria (12 Gram-positive and 18 Gram-negative), makes 330 combinations, of which only 7 potential tests were not performed. (I think the ellipsis means “not performed”.) Each of these 323 tests was done at 3 concentrations, making 969 data points in a single table.

To turn this into graphs would require many graphs. It would also require us to turn the X’s and +’s into a semiquantitative system (let’s say + = 4, ± = 3, X = 2, —* = 1, and — = 0). And since each data point is restricted to those 5 possibilities, you wouldn’t gain much by looking at a bunch of individual dots or bars anyway.

Let’s leave it as a table.

The main flaw of this table is that all the symbols look similar, except “—” . The bottom half of the table is a wall of symbols for different degrees of growth. It’s hard to see trends, because the symbols are hard to distinguish from each other, so it looks like every bacterium is resistant to every drug. Which is not quite true.

Instead of these text symbols, how about something that may be more intuitive — representing the 5 levels of growth as 5 colors. This may not be intuitive in every culture, and it may not work for the color-blind or in B&W printouts, but I took the liberty of changing the table. Now as bacteria proliferate, they pass from “no growth” (white) through yellow, green, and purple stages until they attain “growth like control” (charcoal grey). At first I wanted to range from white to black, but if the cells are black you couldn’t see the lines between them. Or maybe you can’t see them anyway.

For the tests that weren’t done, I replaced the ellipses with neutral grey. And finally, the two types of “diphtheroid bacilli” produced exactly the same results, so they were conflated into one.

dyestuffs-table-1

So where does this data lead us? Krumwiede and Pratt draw limited conclusions. The tables speak for themselves. To briefly summarize their Summary section: the “streptococcus-pneumococcus group” is more dye-resistant, and the “dysentery bacillus group” is highly unpredictable with dye-resistance showing “no correlation with the common differential characteristics”. Also, they discovered mutations that make bacteria lose resistance. (“Among Gram-negative bacteria a strain is occasionally encountered which will not grow on violet agar, differentiating it from other members of the same species or variety.”) Finally, in the middle of the Summary section they say this.

The reaction is quantitative, although the quantitative character is more marked with some species than with others.

Now, a modern reviewer would look unkindly on that sort of admission. First, it seems like it’s not quantitative, it’s semiquantitative. Instead of measuring the number of colonies, you’re grading “growth” on a scale from 1 to 5. And which are the species that don’t have a “quantitative character”? Why don’t you use some other method to see how well those ones are growing? And by the way, how repeatable is your eye test? And what does it mean exactly? Let’s see examples of “growth like control”, “restrained growth”, and “markedly restrained growth”. Does it mean there were fewer colonies, or the colonies were smaller, or both? And why use the same 3 concentrations of all 11 drugs? Maybe 1:500,000,000 would have still killed the bacteria, and be less toxic to patients.

That’s what I’d say, if the journal hadn’t told me “Your review is now 100 years late and consequently is no longer needed.”

* * *

One more paper: from 7 years later, Gay and Morrison in the January 1921 Journal of Infectious Diseases. Whereas Krumwiede & Pratt (1914) was the first in their series, this is a sequel.

gay-morrison-title

Krumwiede & Pratt took a limited range of dyes, and used then on every sort of bacteria they could find. Now Gay & Morrison use every dyestuff they can find on a limited range of bacteria. This is a much longer paper, so I’ll just address their first set of data, about bacterial growth in culture, and ignore their innovative rabbit empyema model.

Table 1:

gay-morrison-table-1

In this table, the numbers mean the reciprocal of the minimum inhibitory concentration (MIC). The MIC is the smallest concentration of the drug that will prevent bacteria from growing. If the MIC is 5 ng/ml, the bacteria should grow if the concentration is lower than that, and the bacteria should die if the concentration is 5 mg/ml or greater.

“2,000” means a 1 to 20,000 dilution. I think that’s weight/volume (1 gram in 20,000 milliliters). The lower the number in this graph, the more concentrated the drug needs to be to kill bacteria. A drug marked “2,000” needs to be a thousand times more concentrated to have the same effect as a drug marked “2,000,000”.

This table is clear. Intuitively we look at the numbers and recognize that 2,000 is smaller than 20,000 and therefore represents less of a dilution. If they were in scientific notation, it wouldn’t be as intuitive.

The only problem is the use of “0”. The most concentrated dyes they used were at a 1:2,000 concentration. If the bacteria still grew, they listed this drug as “0”, or not active against the bacteria. Taken literally, “0” means Staphylococcus would grow on media consisting entirely of methylene green. I would just write the word “inactive” instead of the number 0.

* * *

I might also expand the table to include the rest of the 40 dye molecules Gay & Morrison used in this study. That’s right — in addition to the 12 molecules listed above, they have data on Acid fuchsin, Acid violet, Acridine orange, Azo-acid red, Basic fuchsin, Benzo-azurin, Brilliant cresyl blue, Columbia blue R, Congo red, Crystal ponceau, Cyanin B, Diamil blue, Erioglaucin A, Janus dark blue B, Methylene blue (medical purity), Methylene blue GG, Neutral red, New fast green 3B, Nile blue, Oxamin violet, Rhodulin violet, Safranin, Sauer grün, Scarlet 6R, Setocyanin, Sulphon acid blue R, Toluidin blue, and Wasser blau.

But most of the data is in paragraph form. And it’s not immediately clear.

gay-morrison-excerpt

Let’s make an expanded version of the table above, containing all the dyestuffs. Even the dyestuffs that never killed any of the bacteria. And make some other changes:

  • Keep them arranged in order of effectiveness, but put the most effective ones at the top.
  • In fact, divide them into categories. Those that inhibited all 3 bacterial species, those that inhibited 2, those that inhibited 1, and those that were completely ineffective.
  • Update the nomenclature. We consider Bacillus typhosus to be Salmonella now.
  • And… why not color-code the chart. Maybe blue dyes will do one thing, and red dyes will do something else. Might as well include that information. Patterns may emerge.

gay-morrison-table-2

Isn’t it nice to see those colors? And it shows how much easier it is to kill Streptococcus pyogenes. There’s only one dye that kills either Staphylococcus or Salmonella but fails to kill S. pyogenes. And it shows that blue and red dyes aren’t very antibacterial, while green ones kill bugs dead. Why is that?

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Gallery: The amazing ink-proof yeast capsule

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

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

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

* * *

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

crypto-benham

* * *

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

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

crypto-duryea

* * *

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

crypto-carnecchia

* * *

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

crypto-schupbach

* * *

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

crypto-love-1985

* * *

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

crypto-bottone

* * *

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

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

crypto-neill

* * *

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

crypto-littman

* * *

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

crypto-bulmer

* * *

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

crypto-dykstra

* * *

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

crypto-chang

* * *

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

crypto-doering

* * *

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

crypto-zaragoza-2d

* * *

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

crypto-zerpa

* * *

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

crypto-zaragoza-3d

Data update: Destroyed by freezing does not equal alive

In the experiments reported in the present paper a number of active agents, some undoubtedly living, others equally unquestionably not living, and still others of a doubtful nature, were subjected to repeated freezing (-185°C.) and thawing. By these tests it has been possible to determine that mere destruction or inactivation of a substance cannot be accepted as proof that it existed in a living state.

In 1926, legendary virologist / bacteriologist Thomas M. Rivers addressed the still-extant question “Are viruses alive?” through the simple experimental method of freezing them over and over. The paper in question came out in the Rockefeller Institute house publication Journal of Experimental Medicine (vol.45[1]: pp. 11-21). Not exactly a gripping title.

repeated-freezing-rivers

What kind of data is in here? Well, for most of the graphs they take an “active agent” (either bacteria, virus, or enzyme), freeze-thaw it “as often as desired”, and then see if it’s still active. The data is pretty straightforward, with a separate section for colon bacilli, a section for Virus III, a section for “a bacteriophage lytic in colon bacilli”, and so on. But it’s never summarized in a way that compares the different “agents” to each other. Let’s try to do that.

The low temperature (-185°C.) used in the experiments to be reported was obtained by means of commercial liquid air which was transported from the plant to the laboratory in Dewar flasks. Desired amounts of the air were transferred to deep Dewar beakers where small amounts of the substances to be frozen, enclosed in Noguchi tubes, were completely immersed for several minutes. After the substances had been completely frozen they were quickly thawed in tap water (16-18°C.).

What does “active” mean for these various substances? How did they determine that freeze-thawing had destroyed the substance’s activity? This was different for each substance.

rivers-activity

Bacteria can be measured the same way we measure them now, by making serial dilutions and plating each dilution on agar, then seeing how many colonies grow. Bacteriophage can be measured the same way, by first making a “lawn” (agar plate fully covered with bacteria) and applying different dilutions of bacteriophage, then seeing how many “plaques” (holes in the lawn) are formed by the phage killing the bacteria.

Complement can be measured by seeing how long it takes to destroy “given amounts of red blood cells in the presence of a great deal of amboceptor“.

Trypsin can be measured somehow, Dr. Rivers doesn’t specify except by saying that his colleague Dr. Northrop took care of that part of the study. Nowadays Dr. Northrop would be a co-author. But the paper would then have to list Rivers as both the first author and the last author somehow, since Northrop didn’t do enough to merit either status.

Anyway, to find “details of the technic” we are directed to a paper by Northrop and Hussey (1923), showing a very clever method by which a solution of gelatin is exposed to trypsin, and at different timepoints the gelatin’s viscosity is measured.

northrop-graph

And how do you measure viscosity? With a viscosimeter.

northrop-viscosimeter

I’m having trouble understanding sentences like “The gelatin-water time ratio was approximately 3”, but the point is that you can measure the amount of trypsin by measuring how fast it turns gelatin into runny gelatin. Nowadays you would use a colorimetric assay, in which trypsin cuts the protein that has some sort of colored label attached to it, and you would measure how much colored label gets released into solution.

* * *

Finally, the three mammal viruses.

“Vaccine virus” and “Virus III” are both introduced to the skin of rabbits, probably by scarifying and then rubbing the virus into the scratches. They look for a “virus reaction” in the skin surrounding where the virus was inoculated, and measure this semi-quantitatively based on how bad of a sore forms. I hope they aren’t simply measuring the immune response to the inoculation, because even killed virus should produce some immune response.

“Vaccine virus” is basically what we now call vaccinia virus. “Virus III” is a more interesting question.

All stocks of Virus III were lost some time before the invention of electron microscopy. Nobody can now be sure what exactly this rabbit-specific virus was, but it was probably Leporid herpesvirus 2, as described by Nesburn in 1969. For an objective summary of the Virus III story, read the page on LHV-2 (p. 380) in The Laboratory Rabbit, Guinea Pig, Hamster, and Other Rodents (Academic Press, 2012).

“Herpes virus” (Herpes simplex) activity is measured differently. They inject HSV into rabbit brains, and they look for dead rabbits. It seems like this assay should actually be quantitative, based on looking at how long it takes the rabbits to die, but all their rabbits either died within a week or lived longer than a month, so the results were clear without measuring time to death.

 * * *

The results are pretty simple, which is why I would like a summary figure instead of a series of tiny individual tables for each thing they studied.

rivers-summary

Some comments on the data:

  • “Locke’s solution” is like what we call Ringer’s solution, the intravenous fluid given to people who have suffered blood loss. This page indicates that it’s the same as Ringer’s solution, but buffered by bicarbonate instead of lactate. But most other sources indicate that it also includes glucose, making it closer to Tyrode’s solution.
  • Locke’s solution is basically a physiological salt solution, so what’s the difference between that and the “physiological salt solution” used in the phage experiments? The latter doesn’t contain glucose, and isn’t buffered; instead of a pH of roughly 7.6, the pH is “between 6 and 7”.
  • Vaccinia virus was the most stable, retaining the ability to create a skin lesion even after 34 freeze-thaws. Virus III was destroyed after 12, and the other herpesvirus was destroyed after 24.
  • Both bacteria and bacteriophage had more than 99% of their activity destroyed by freeze-thawing in Locke’s solution. All three big viruses were more hardy than that.
  • I don’t know what a “1:10 dilution of trypsin” is. How many USP Trypsin Units is that? What’s the molarity? We have to take Dr. Northrop’s word for it, that it’s the same system for trypsin dilution he always uses.
  • At a mere 1:10 dilution, complement and trypsin were not damaged by 12 freeze-thaws. But when diluted further, they were very susceptible to freeze-thaw.
  • In fact, bacteria and bacteriophage were also more susceptible to freeze-thaw at low dilutions. Even vaccinia virus lost all its effect after 34 dilutions at 1:100,000 dilution, although it didn’t have much activity at that dilution to begin with.
  • Of further interest: the difference between high and low dilutions was only observed in Locke’s solution or salt solution, not broth. Remember these cutting-edge findings when you make your own aliquots.

Viruses can be RE-activated by light?

When you’re always looking at old sources, you run the risk of condescending to the experts of the past, who believed scientifically plausible things that now seem obviously wrong. More than once I’ve been ready to point out some amusing practice of the distant past, only to find out that it’s a perfectly valid fact that I (having no medical or physiological training) had never heard of.

One example is “vicarious menstruation”. Is it possible that menstruation could manifest as a nosebleed, or sores in the mouth (sometimes called “herpes”)? Or as a pair of ulcers on the legs, as D.H. Galloway of Roswell, New Mexico reported in 1913? Isn’t it more likely that these stories are exaggerated, or are coincidental? But yes, some combination of hormone levels and blood pressure creates that phenomenon in some women.

galloway-vicarious-menstruation

Another one is “activated milk”, which contained a substance called “viosterol” that was in high demand for preventing rickets in children. Activated milk? Activated by what?

Ultraviolet light, it turns out. Did this work? Well, UV light turns cholesterol into vitamin D3 when our own bodies are exposed to the sun, and it turns the fungal (yeast) equivalent, ergosterol, into vitamin D2. Cows could be fed UV-activated yeast to make them produce “activated milk”, or activated yeast extract could be directly added to milk. Either of these was a way of “activating” milk that probably worked. Exposure of normal milk to UV light seems like it would be a waste of time.

Whether its benefits were exaggerated or not, activation of milk and other foods was extremely popular, as described in Michael Holick’s great historical review in Public Health Reports, called “The Vitamin D Deficiency Pandemic: a Forgotten Hormone Important for Health”. The drug and food industries fought over whether companies like Fleischmann’s Yeast could claim their products were the equivalent of vitamin D supplements. Here’s a contemporary excerpt from Cartels: Challenge to a Free World, Wendell Berge’s 1944 classic of vaguely paranoid economics.

cartels-activated-milk

And all the way into the 21st century, there’s heated debate over whether vitamin D2 (the vitamin D in most supplements) is an appropriate substitute for our own vitamin D3.

* * *

So anyway, here’s another real thing that looked weird and debunkable at first glance.

In virology papers from the fifties and sixties, there are many mentions of something called “photoreactivation”. This started with 1949 work by future Nobel laureate Renato Dulbecco, done in the Indiana University laboratory of future Nobel and National Book Award laureate Salvador Luria. In 1950 Dulbecco summarized the story.

Kelner (1949), working with conidia of Streptomyces griseus, discovered that light belonging to the visible range is capable of reactivating biological material that has been rendered inactive by ultraviolet radiation (UV). Shortly after Kelner’s discovery was known, a similar phenomenon in bacteriophages (bacterial viruses) was observed by accident. Plates of nutrient agar containing UV-inactivated phage and sensitive bacteria had been left for several hours on a table illuminated by a fluorescent lamp. After incubation it was noticed that the number of plaques was higher on these plates than on similar plates incubated in darkness. A short report of this phenomenon of “photoreactivation” (PHTR) has already been published (Dulbecco, 1950).

We’ve been using UV light, gamma rays, and chemical agents like nitrogen mustard to make “killed” versions of viruses, safe for use in vaccines. And now it’s possible that visible light could then re-activate these menaces? Should vaccines be stored in the dark?

Beyond  bacteriophages, many other viruses were found to be capable of photoreativation. A sample:

  • 1955: “Of the three viruses we studied earlier, tomato bushy stunt and the Rothamsted tobacco necrosis virus showed the phenomenon of photoreactivation, and tobacco mosaic virus did not … Of the six viruses that did [in this study], potato X showed it much the most strongly, tomato bushy stunt and a tobacco necrosis virus the least; cabbage black ringspot, cucumber mosaic and tobacco ringspot were intermediate.”
  • 1958: “Thirty minutes of illumination at 300-380 f.c. gave substantial photo-reactivation [of] potato virus X”
  • 1961: Tobacco mosaic virus particles can’t be photoreactivated, but RNA preparations from the virus can.
  • 1967: “Photoreactivation of UV-irradiated blue-green algal virus LPP-1”
  • 1967: “By contrast, photoreactivation of the irradiated [tobacco necrosis virus] was observed in French bean and tobacco, but not in Chenopodium.”
  • 1968: Pseudorabies virus can be photoreactivated in chick embryo cultures, but not in rabbit kidney cells.

In the last of those quotes, it’s becoming clear that viruses don’t photoreactivate on their own. They photoreactivate inside cells. You can use UV light to damage the DNA (or RNA) of a virus so it can’t multiply. But it may still infect cells if the protein coat is intact. Then once the viral DNA (or RNA) is inside the cell, the cell’s DNA repair mechanisms can go to work. One of these is photolyase, found in plants, bacteria, fungi, and some animals, but not mammals. Blue light activates this enzyme to reverse the DNA damage caused by UV light (specifically, covalently-linked pyrimidine dimers).

So instead of thinking of photoreactivation as something that happens to certain viruses, we should think of it as something that happens in certain types of cells, to viral DNA as well as cellular DNA.

By 1958, Dr. John Jagger (who does not have a Wikipedia page, though his wife, also a scientist, does) was already able to write a fantastic review of photoreactivation in general (not just viruses and bacteria), saying:

Photoreactivation seems to be possible whether the UV damage occurs in the liquid or the solid state. However, the reactivation seems to require not only the liquid state, but a rather complex environment, similar to that within a living cell.

It doesn’t quite require a living cell, but it requires “cellular material”. A cellular extract still contains the photolyase enzyme.

* * *

You’ll notice that the above examples are almost all plant viruses. This is partially because plants were a very convenient system for virology in the era before cell lines, but it also has to do with the importance of light in plant biology. Dependent on the sun, they need to be able to counteract the negative aspects of ultraviolet light.

But it’s also clear that photoreactivation takes place in insects and fish.

The data show that fish cells have an efficient photoreactivation system at wavelength > 304 nm that reverses cytotoxicity and dimer formation after exposure to filtered sunlamp irradiation of a shorter wavelength (lambda > 290 nm). Shorter wavelengths in UVB (> 304 nm) are more effective in photoreversal than longer ones (> 320 nm). As a consequence, 50-85% of dimers induced by these wavelengths in fish are photoreactivated while they are being formed. A major cytotoxicological lesion is the cyclobutane pyrimidine dimers. Cultured human fibroblasts do not possess such a repair system.

What about that paper above, in which chicken embryo cells enable pseudorabies virus (a herpesvirus) to reactivate? That looks weird, to me at least. Shouldn’t chickens, being warm-blooded animals, be grouped with mammals rather than fish? But chickens aren’t mammals. This table, from Photoreactivating-enzyme activity in metazoa [Cook JS, McGrath JR [1967] PNAS 58(4):1359-1365] sums it up.

photoreactivation-in-metazoa

Mammals have other DNA repair mechanisms, but we lack photolyase. Which as it turns out, makes us kind of weird.