Friday, 25 November 2016

Gearing up the Gram stain

In my Yr3 Food and Fermentation Microbiology course aka F&F, the kids do a helluva lot of Gram stains. This chemical /microscopic test usefully divides the bacterial world into two enormously variable classes based on properties of the cell wall. That's important because the cell wall is a very common target for our 20thC anti-biotics some of which only operate against one class or the other. Penicillin, for example, used to work against 'Gram-positives' like Staphylococcus, Streptococcus, Clostridium and Listeria and not-so-much against enterics like Salmonella. Of course penicillin does not work against anything anymore because of the rise and rise of anti-biotic resistance - MRSA anyone? Because resistance has been promoted by flaithulach use of anti-biotics, no medico wants to prescribe an anti-biotic which will be ineffective against the cause of the infection while promoting resistance among the microbial innocent bystanders. So one of the first tests in the Path Lab is a Gram stain.

Whom do we have to thank for this work-horse of bacterial diagnostics? Why, Dr Gram of course. Hans Christian Gram, a Danish citizen, was working  in the city morgue in Berlin and used much patience and ingenuity to develop a stain that would make bacteria more visible under the microscope when he was trying to establish cause of death (often TB at that place and pre-antibiotic time). The fact that half the bacteria he encountered stained blue-purple and half pink [see R, if you look carefully you can see that the blue ones are spherical and the pink are rod-shaped - if you can't look carefully under pixellating conditions you're not cut out for microscopy] was a by-product for him but super-useful for pathologists - and F&Fists - ever since.  Gram must have been patient and ingenious because we have finished up with a protocol that involves fixing, staining, washing, different staining, decolourising, washing, counter-staining, washing, drying, viewing.

Every year we get a leaven of French students in Yr3 under the Erasmus scheme. These étudiants formidables are really good for us because they are different. And formidable because les lourdauds et monoglots will have stayed at home in Nantes or Paris. In Ireland the protocol is carried out by flooding the glass microscope slides, the this then that, on racks above a sink or staining tray and letting the excess stains run off down the drain. In France otoh they keep the stains in special glass jars and transfer the slides from one to the next in order.  Every week or so, they dump the stain and make up fresh, I don't know which is more wasteful but the french method seems more efficient and less messy. One issue, I suppose, is whether you are doing one preparation or many-at-once.

Last Tuesday I was being adult-in-the-room for the Yr4 research project students. Students are not allowed in any lab unless supervised. Now, my level of expertise at the bench doesn't make me the most competent person in the room to give a tutorial on how to use a Spectrophotometer but after 40 years in science I'm not totally useless with my advice. One of the kids had a pile of Petri dishes and was fixing a sample to a slide prior to doing a Gram stain. This involved holding the slide with a pair of wooden tongs and passing it through the flame of a Bunsen burner to dry off the water and bake the bacteria unto the glass. You have to be a little slippy here, because a beginner's error is to crack the slide in too hot a flame - it's one of the reasons for wearing safety-glasses in the lab. Looking at the mountain of Petri dishes to be processed, I realised >!shazzammm!< a more efficient way of drying/fixing multiple samples. Why not, I suggested, spread the soup on several different slides and leave them on top of the hot-as-hell (well, a tad over 100oC aNNyway) autoclave to dry off. That way a) no cracking b) you can prep-up the next batch while waiting.  And surely 25x samples is the time to do the Gram stain à la mode français? More time for tea that way.

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