so what do u do when you're sick and just want to buy some fish antibiotics to go on ur merry way? u test to confirm ur suspected affliction... what are my symptoms? sore and swollen throat and lymph nodes, fever, swallowing pain, no cough... our suspected enemy is streptococcus pyogenes, a Group A β-hemolytic, catalase-negative, gram positive bacterium--aka strep motherfucking throat... let's test it out. take a throat swab. slab it on a slide. stain it with crystal violet, a + charged dye which seeps into the - charged bacterial cell walls and membranes. wash it off, then hit it with iodine. it'll differentiate between gram +/- bacteria by forming a complex that'll encourage gram + bacteria to hold on to the dye in the peptidoglycan layer. rinse the excess away with denatured alcohol, then a bit with water. at this point, gram - bacteria should be decolorized since it can't hold onto the crystal violet-iodine complex. flash dry with ur favorite lighter, put a cover slip on it, then place it under a microscope. throw it on the lowest mag, then pan around until u see some clusters. >zoom.png, then once ur at 100x mag and if ur lens supports it, add some immersion oil. if u can't get a definitive answer by checking out the appearance (small spherical chains), it's time to culture it.
culturing it is a whole other thing. when u have a live sample, the number of bacteria that are gonna be observable with a simple stain is gonna be low. also, ur gonna be looking at skin cells and other bacteria at time of collection--u want a clean view. ideally, you'd like to isolate the specific bacteria you'd wanna look at, so u take a swab again, and streak it on a petri dish with some nutrient agar. u put the petri dish in an incubator for a day or two. u then plate it on some sheep's blood agar, the preferred medium for culturing strep bacteria. u culture it for a few days, and u should see some colonies. ur also testing for beta hemolysis (complete destruction of red blood cells) here, which means u need to see a clear radius around the streaks on the petri dish indicating that the bacteria's been producing an exotoxin called hemolysin, which destroys the red blood cells. once u have some colony growth after a day or two, identify it based on its morphological and biochemical properties. u then go on ur merry way. BUT WAIT! gene, i can't get any sheep's blood agar. i don't know any sheep, let alone even like me enough to let me have some of their blood. what do i do?
in mid 1900's, a dude named Oswald Robertson started reporting about better blood transfusions near the front lines in France during World War I. you get shot, you lose blood, tons of dudes were getting shot and there wasn't enough blood to go around, so they needed to get some blood flewed in, but it wasn't really feasible to do for a while since blood was hard to keep. some dudes in the late 1800's (Hammarsten in 1875, Arthus & Pagés in 1890) started figuring out that calcium chloride would keep blood from coagulating as fast and fibrin levels low but they had no fucking idea why it worked. other guys tried using oxalate, an ester of oxalic acid that's found in fucking spinach, but it was a bit risky on the kidneys in high enough dosages. didn't stop that binch from trying anyway on DOGS, multiple times bro. it's said though that "the dogs appeared to be none the worse for their experience", holy shit. anyway, shortly after, they finally figured out that it was citrate that prevented clotting after testing 90cc of blood mixed with 10cc of a 5% solution of it (spoiler: it was because of the affinity of calcium to citric acid). like a year later, a french dude (fuck) finally made it clear and told his friends it stops coagulation because it reduces the concentration of ionized calcium below the level needed to form clots. bitches kinda just took this and ran with it just pulling blood out the jugular in rabbits (Todd & White, 1911) then hitting it right back in another rabbit after citrating it. then FINALLY in 1914 (Hustin), they started human transfusions. dude ran some experiments, found that 0.2% citrate stops blood from clotting for 30 mins, but then found out later that red blood cells were having a harder time getting oxygenated, so he tried sodium chloride, which did way better. dudes kept trying for years afterwards with different combos of various chemicals and compounds to try to reduce clots and increase storage time, but honestly nobody had much of an idea about what the other guy was doing. enter: 'qui retarderait également' coagulation using a glucose-saline solution.
ok, so by now we've figured out that sodium citrate helps stops blood from clotting, but we still needed a better way to preserve it if we were gonna carry that shit the long way. Rous & Turner (1916) thought red blood cells don't let any sugar through, so they might work as colloids to keep the blood in suspension (they were kind of right, sucrose doesn't get through but glucose did. still, we're getting somewhere!!). glucose and sucrose ended up being the best candidates to prevent cells from getting murked while being stored. >keep_trying.jpeg and waow, citrate-sucrose will keep blood for TWO WEEKS. still, glucose ended up being the better option because less red blood cells died off in storage for longer periods of time (a fucking MONTH!!??!), so they took this shit to the british military hospitals in france. they still had to siphon out some of the supernatant (a clear liquid with blood cell lysis that floats to the top) and filter the residual clots through gauze. not a super refined and demure process, but it worked. for bloodthirsty wartime, this shit was pretty much a godsend for long-er distance blood transfusions and storage. and now we get to use it because we're broke and curious. my, how times change.
some of these may not be readily available to view. i recommend going to uhhh anna's archive and dropping the DOI ehehe
- my experience in a microbiology lab
- https://pubmed.ncbi.nlm.nih.gov/30943176/
- http://www.thelabrat.com/protocols/ACD.shtml
- https://journals.asm.org/doi/10.1128/jcm.02631-05
- https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2953122/
- https://onlinelibrary.wiley.com/doi/10.1046/j.1365-2141.2000.01827.x
thanks for reading girlies, i'm in atlanta right now and it's 3 5 am but i've held off writing this for too long lol