Richard Phillips Feynman (May 11, 1918 – February 15, 1988) was an American theoretical physicist. In this beautiful video he explains fire, how plants come from, and photosynthesis and brings it all back full circle. He breaks down everything with such clarity that it’s obvious this is his passion.
Did you know there are bugs flying not just around your home but thousands of feet high in elevation? And not just some but BILLIONS of them?!
Check out this incredibly informative video. It starts slow for the first minute but get suuuper interesting after!
Pharmacology refers to drug administration (including dosage) and distribution in the body (pharmacokinetics). The area under the curve includes T half-life, peak concentration, time to achieve peak, elimination and protein binding. It’s one of the most important aspects of drug study, including antibiotics.
Peak and Trough
Let’s look at the peak and trough of gentamycin, which is an aminoglycoside. If the concentration is too high, the peak could lead to ototoxicity and nephrotoxicity. So it’s essential you measure peak and trough. If the peaks are exceeded, their middle ear apparatus are destroyed so they can’t tell their position and they could have kidney failure.
The normal peak and trough are 10-12ug/ml and 1-2ug/ml, respectively. The blue line shows the appropriate administered dose while the black line shows one that is excessive.
The route of administration is determined by the site of infection, the level of antibiotic required at that site, and the patients status and the urgency of appropriate treatment.
- Intravenous (IV) administration typically gives the highest concentrations in the shortest time and is thus preferred for seriously ill patients
- Intramuscular (IM) administration produces lower concentration levels over a longer period than IV but a higher concentration than oral administration.
- Oral administration is beneficial, dependent on the infection, for maintenance doses.
- Other methods of administration include sublingual, mucocutaneous (such as eye, nose, ear) and topical.
For seriously ill patient with SIRS and a gram positive gram stain from the C&S, what route of antibiotic administration do you recommend?
IV because it’s the fastest possible.
Bioavailability affects the concentration of availability of the active drug in the systemic circulation because the drug must succumb to the gastrointestinal tract. It’s also influenced by solubility, which relates to the excipients (vehicle; tablets and capsules for oral, suppositories for rectal) in which the material is in. In regards to the question of generics, the FDA regulates the generics so they are the same as the proprietary versions (at least for those manufactured in the USA).
An example of bioavailability are the Fluoroquinolones (Ciprofloxacin, Levofloxacin and so forth). It’s recommended that you should avoid taking it 2 hours before a meal or 4 hours after a meal and also avoid antacids.
LOOK AT PAGE 493 FIGURE 21.3.
Drug distribution in body tissues should be considered in pharmacology/pharmacokinetics. IM/IV/Oral, it doesn’t matter, the drugs all get to the blood/systemic circulation. Even the nasal cavities are highly vascular and it has been shown (chinese showed this for centuries, long before jenner formulated it). Sometimes, you want the drug to be concentrated in the lungs sometimes, but with a pill taken by mouth, it has to cross into the blood stream. If it’s bound to protein, it can’t cross because the sheer size of it prohibits crossing. This is something the drug manufacturer has to document that protein binding is not significant or there is some mechanism where the drug can be freed by the protein.
If the drug binds to protein it will not cross into the tissue. (In other words, protein-bind = cannot cross)
Not protein bound (unbound drug) means it crosses by…
- dissolving in lipid (lipid soluble drugs) because the cell membranes are made of lipid.
- protein pores which are transport proteins
- means of enzyme catalyzed transport
One of the quintessential antifungals: Amphotericin B, used widely for systemic fungal infections, is extremely toxic. And why would that be an issue? Both fungi and your cells are eukaryotic. In the business this is known as amphoterrible. Some patients rather die than take it because if they do take it they get this metallic taste in their mouth, persistent ear ringing, nausea, upset GI and vomiting.
Part of the toxicity can be overcome and reduced by targeting delivery. Amphotericin B is now encapsulated in liposomes (now known by the market name: ambisome). A liposome is a fat globule (lip=lipid=fat), and inside of it is amphotericin B. It will dissolve in the lipid membrane due to the similar structure and that will ensure that the delivery to the tissues is much more facile rather than circulating ad libitum (freely) throughout the body. Amphotericin B is now marketed as Ambisome because it’s in this lipid vesicle.
Reference Page 494: Read “Drug Distribution” section to get a fuller idea.
Ciprofloxacin has an extended preparation of cipro. There are several coatings so there’s an extended release of the drug (by dissolving the layers slowly), that allows a different dosing regimen.
Spectrum of activity
Antibiotic: Only refers to drugs that are effective against bacteria.
Antimicrobial: Includes antiparasitic, antifungal, antiviral. An antiviral drug is commonly referred to as an antibiotic but it’s not an antibiotic.
The range refers to the number and variety of organisms that can be covered. When we talk about any antimicrobial agent, we talk about whether it’s a broad spectrum (covers a wide range of organisms) or narrow spectrum (covers a narrow range of organisms; generally one type of bacteria).
Turn to page 495, figure 21.5: YOU MUST KNOW THIS CHART. Know which antibiotic is used against what type of bacteria
Pencillin: When asked for a narrow spectrum antibiotic on the test, DO NOT say penicillin, as it can be used against outside of its spectrum of gram positive activity, even though the table in the book says it’s a narrow spectrum. Ampicillin is a variation of penicillin. Up to 15-20 years ago, penicillin was the treatment of choice for N. gonorrhoea and it’s not anymore due to resistance.
Isoniazid/Ethambutol: Narrow spectrum, effective against mycobacteria.
Polymyxin B: Narrow spectrum: effective against gram negative bacteria.
- Ampicillin (covers gram positive and gram negative)
- Chloramphenicol/tetraycycline (covers everything except mycobacteria)
- Streptomycin (covers everything except gram positive)
What is the significance of using broad versus narrow?
1. Broad spectrum gives greater perturbations to normal flora so it leads to secondary infections (pseudomembranous colitis; antibiotic associated colitis).
2. The possibility of resistance will be increased with broad spectrum because it’s used against a wider variety of organisms. In other words, the chances of resistance developing are more likely with the broad spectrum antibiotic because it covers such a wide range of organisms so there’s a greater potential for resistance.
Some antibiotics are classically associated with this: Clindomycin, for example, in the early days had an unsavory reputation for an increased incidence for pseudomembranous colitis and it was particularly useful against anaerobic bacteria. The broader the spectrum, the more likely it is to perturb normal flora.
You will be asked a question on fecal transplant (aka fecal bacteriotherapy):
- Fecal transplant restores the colonic flora by introducing healthy bacteria.
- The procedure involves single to multiple infusions (by enema, colonoscope, etc) of bacterial fecal flora from a healthy donor, into a patient suffering from Clostridium difficile infection (pseudomembranous colitis; antibiotic associated colitis).
Resistance to Antibiotics/Antimicrobial agents
Factors influencing resistance:
- Medical “Malpractice” (overuse). Not deliberate but an overuse.
- Cross border / Internet access / International access without prescription.
- Questionable formulations(preparation offshore) / Counterfeits
- Self medication
- Rampant & aggressive advertising by “BIG pharma.”
- Increased patient demand and access.
- FDA pressures. (Not so common with antibiotics but rich young white professionals, like doctors, lawyers, pressure the FDA to fast track the medications w/o adequate research and the data may be soft.)
- Antibiotic residues in meat/fish. Xenobiotics can be hormonal-like, and in some countries like Brazil you have early onset of puberty.
- Animal husbandry. (supplementing cattle, feed with antibiotics & hormones..etc. Cattle put on more weight and they can make more money)
- Poor patient compliance (Premature discontinuation or missing a dose; Certain levels are required for efficacy)
- Access and availability
- Insurance coverage, which also affects availability. (It’s not your doctor that decides what drug you get, it’s your insurance). A social disaster.
- Migration / International Travel / Offshore medical treatments. The latest denizen on the block, Extended spectrum organisms.
- Medical tourism (TNA: titties and ass in India).
- Continued adaptive responses, mutations, changes in the target organism.
Bacteria can adapt so quickly to changes, it surpasses anything we are capable of. Every single antibiotic discovered to date has been shown to be associated with resistance in target organisms, even in circumstances where there is no evidence of prior exposure.
Since bacteria have such a wonderful adaptive capability and can multiply so rapidly, that one in a million or billion chance creates naturally occurring resistant organisms. You start off with a mixed population of resistant and susceptible organisms. You treat with antibiotics and the susceptible ones will be eliminated and you’re left with a resistant population which will multiply like crazy and that drug no longer become effective.
For this reason, the CDC launched an antibiotic tracking system for hospitals. Antibiotic use can lead to antibiotic resistance which can increase the risk of hospitalization or lead to treatment failure in sick patients, according to the CDC.
Antibiotic-resistant infections are likely to require longer and more costly hospital stays and have higher mortality rates. If first-line drugs don’t clear up the infection, second or third-line drugs may be even less effective, more toxic, and more expensive.
Real threat of returning to a pre-antibiotic era: Drugs no longer useful because organisms are resistant to everything we have come up with.
With every single antibiotic noted to date, within 6 months of use, antibiotic resistance has been documented. So there’s no antibiotic which bacteria have not developed resistance. It’s microbial survival of the fittest.
*T/F Question! – Emerging resistance to N. Gonnorhea is developing. (true)
Basic mechanisms of resistance
One single organism may have several different mechanisms to several drugs. The bacteria can either destroy the drug or alter the drug or inhibit the drug or do all of them.
Reference: pg. 496 Table 21.2
This isn’t an exhaustive list, there’s many variations of each theme. Know the 3 primary mechanisms and one antibiotic associated with each one. These are all ways that bacteria can destroy/alter/inhibit the drug (antibiotic).
- Destroys. There are several ways.
- Plasmids can encode enzymes that chemically alter aminoglycosides (by acetylation or phosphorlyation)
- Plasmids can encode beta-lactamase which inactivate beta-lactam antibiotics such as penicillin and cephalosporins.
- Alters drug.
- An aminoglycoside normally tries to bind to a 30S ribosome subunit, so as a resistance mechanism, the bacteria makes a different, altered ribosome that won’t allow the drug to bind to it and won’t be inhibited.
- Quinolone normally tries to bind to DNA topoisomerase to inhibit DNA synthesis but the bacteria could make an altered DNA topoisomerase so the antibiotic won’t bind to it.
- Rifampin normally binds to RNA polymerase to inhibit RNA synthesis but the bacteria makes an altered polymerase so it won’t bind to the drug.
- Inhibits drug entry or removes drug.
- Penicillin binds to proteins in the outermembrane to enter the cell and block peptidoglycan synthesis, but the bacteria can change the shape of these porin proteins so the drug can’t enter.
- Erythromycin and tetracycline normally bind to ribosome subunits to inhibit protein synthesis, but the bacteria creates a new membrane transport system to literally pump the drug out of the cell.
Increasing the number of different mechanisms of resistance increases the likelihood of resistance.
T/F Question – Bacteria are conscious or sentient entities. (true)
What evidence is there that bacteria are conscious or sentient entities?
Capsule, spore , nutrition, temperature, adaptive, listeria cold enrichment.
- Capsule formation is determined on nutrient availability. Bacteria respond to nutrient excess by making a capsule which is protective and allows them to attach to tissues to produce infection.
- Spore formation and germination are dependent/determined by environment. When nutrients are limited and water is not available, they will form spores. When conditions are favorable, they will germinate.
- Motility associated with flagella. Taxis (movement) is a directional response. Phototaxis. Magnetotaxis. These are all responses to various stimuli which means they need to be coordinated to move in a certain direction.
- Quorum sensing (we talked about this with biofilms!)
Evaluation of Efficacy
How do you know if antibiotics are going to work? It’s the S in C&S (Sensitivity).
Susceptibility testing (for lab practicum).
- Kirby Bauer Disc diffusion
- R – not effective
- I – when there’s evidence of accumulation of organism in blood/bodily fluids.
- S – use
- MIC, MBC -> Broth Dilution
- Agar Dilution
- “E” Test
Question on exam You have a plate with antibiotics, and you look for the zone of inhibition. This zone is arbitrary and has no significance until you refer to the NCLS Chart. If the chart says that 12mm or more is equivalent to susceptibility, whereas 10 or less is resistant, and you have 12 or more, it means it’s susceptible and so forth
If control zones are not within acceptable limits or if the identity of the organism is unknown, the data from the test is invalid and must be discarded necessitating that the test be repeated.
A high M.I.C. (minimum inhibition concentration) typically indicates resistance. (on the KB chart) I’ll give you zone sizes, the results of the test and ask you whether it’s R or S or I
Now turn to pg. 500 – dilution of antibiotics.
You have different graded concentrations, all tubes inoculated with the same organism. The tubes with no antibiotics are control. If the control tubes have no growth, they are dead. (HE WILL ASK THIS). 2 mg/mL has no growth, so the MIC is the lowest concentration that inhibits the growth of the bacteria. I could give you the results like this or a chart and ask you where the MIC is.
To determine the bactericidal concentration, take all the negative tubes and put them on plates. If they grow, they are not totally inhibited. The MBC in this case is 8 mg/ml. The MBC is the minimum bactericidal concentration: the lowest concentration that KILLS the organism, established by taking cultures with no obvious growth, to antibiotic-free media and looking for growth. If the subculture in the antibiotic-free media grows, the bacteria were inhibited but not killed; The MBC is equated by those negative cultures that do not grow on the antibiotic-free media, documenting that the bacteria were killed.
Uses/Applications of MIC/MBC Data: This is a guide to the choice of therapy.
1. If the MBC 64x times greater than the MIC, that indicates TOLERANCE which means the antibiotic is not likely to be effective for treatment. You have to use so much of the antibiotic to kill the organism that you exceed the therapeutic ratio or range and you can harm the patient.
2. Efficacy is mandatory data for FDA approval and essential documentation.
3. Comparative efficacy. In other words, the FDA is not necessarily concerned with proving just another drug without a significant advantage, unless it’s a generic, which the data would have been established by the proprietary brand name. So this is one of the major planks in the approval process so they have to demonstrate that the drug actually works.
4. Even more importantly, it monitors the development of resistance, or changes in susceptibility patterns/response.
You’ll typically see:
MIC50 means ½ of the isolates tested are inhibited (which means they are susceptible).
MIC90 means 90% (9/10) are inhibited.
MBC50 means ½ of the isolates are killed by the concentration.
MBC90 means 90% of the isolates are killed by the concentration.
YOU’LL NEVER SEE MBC 100. There’s always going to be an organism that is going to be resistant, given the fact that antibiotic resistant can be documented without prior exposure and within 6 months of introduction of any antibiotic.
If you require an increase of concentration, it’s indicative of an increase in resistance, you need a greater concentration of antibiotics.
↑ [C] = ↑[R] (C=concentration, R=resistance)
In the year 2000, MIC50 = 1ug/ml
In the year 2010, MIC50 = 5ug/ml
Let’s look at ciprofloxacin… If an effective dose is achieved at 5ug/ml, and you have MIC50 but years down the line 50% of your isolates may require 10ug/ml. If you have an organism that requires TWICE the concentration to be inhibited, it means 5ug/ml is not going to be effective anymore. You can’t just double the dose cause then you have toxicity.
Be familiar with why an organism showing tolerance is not effective to that treatment.
Agar dilution: Rather than having a bunch of tubes for the MIC/MBC, the tubes are replaced by agar, with varying concentrations of antibiotic or antimicrobial agent.
Neutropenics. The big problem is neutropenics are treated with antifungal treatments like Nystatin Px but a lot of those patients end up with fatal fungal infections. So the question is… are the deaths due to antifungal failure? In other words, were the organisms resistant to nystatin? The failure of treatment was not due to the resistance of the organisms.. none of the fungi were resistance. The failure was due to the neutrophil count not being increased.
Fungi are not tested routinely for susceptibilities, although several experts are recommending that this be done. For the most part, the responses are predictable. What is done instead is the tests are done in pools of isolates and they are periodically tested with the agar dilution or now they have modifications with respect to instrumentations that allow you to detect growth dependent on the concentrations
The E test is not as widely accepted in America because here’s a bit of jingoism since it wasn’t developed in the USA. It’s slowly gaining acceptance and becoming more routine now.
The E test is useful because you have a lawn of bacteria and you could put several different antibiotic strips that could be vancomycin, penicillin, gentamycin, tobramycin. Whoever came up with this is certifiably genius.
The concentrations of the antibiotics are layered sequentially and sold commercially. You put this strip on the lawn and you end with growth or no growth or a zone of inhibition.
The point of intersection where the point of inhibition coincides with the level marked on the strip is the MIC. The beauty of this is you could test 4-5 different antibiotics separately and you could get break-point MICs. You will indubitably get questions on the exams with this.
The E test, a gradient diffusion method that determines antibiotic sensitivity and estimates minimal inhibitory concentration. The plastic strip, which is placed on an agar surface inoculated with test bacteria, contains an increasing gradient of the antibiotic. The MIC in ug/ml is clearly shown.
Sites of Action and Examples of Antibiotics Targeting PROKARYOTES
Reference page 501, figure 21.10
You see prokaryotes and eukaryotes. There’s a clinically significant difference between the two. This is a prime example. Not only do these differences reflect specificity of action but explain the toxicity associated with antimicrobial agents.
How is toxicity of antibiotics and adverse drug reactions related to the differences of eukaryotes and prokaryotes?
The specificity of microbial action is related to the structural, physiological and metabolic differences that differentiate microbes from the human body.
You must be familiar with ALL of these sites and the relevant antibiotics that target those specific sites. Any part of the bacteria is fair-game.
What antibiotics inhibit the bacterial cell wall?
- Bacitracin (used topically as an antibiotic).
Read the “Cell Wall” paragraph. You only find peptidoglycan in bacteria. These agents have no effect on eukaryotic cells because all eukaryotic cells lack peptidoglycan.
Question on test: What is the treatment of choice for L forms and why? An agent that inhibits protein synthesis (gentamycin) and penicillin that inhibits the reformation of the cell wall. Some L forms can reform the wall, so if they reform the wall, you use penicillin to inhibit the cell wall and the gentamycin to inhibit protein synthesis, so that’s a synergistic example of antibiotics. (Synergy = 1+1=11).
Beta-lactam ring -> Penicillinase (beta-lactamase) -> Produces penicilloic acid, which inactivates the penicillin. The penicilloic acid is also a product that can trigger allergic/hypersensitive reactions.
What antibiotics inhibit the bacterial cell membrane?
- Polymyxin B. You don’t use it as a widespread use of an antibiotic. It’s used topically. Read “Cell Membranes” paragraph. ALL cells have membranes so it’s not an ideal target of drug action but there are some differences between microbial and mammalian cell membranes. Most drugs that target cell membranes are highly toxic when administered systemically.
- What is the basis of specificity of polymyxin? The eukaryotic cell membrane has a different sterol composition that isn’t recognized as well by the polymyxin.
What antibiotics inhibit the Nucleic Acid synthesis?
- Rifampin (RNA)
- Quinolones (DNA)
- FQ = Fluoroquinolones. ie.,
- When you see FLOXACIN in the drug word, that’s a Fluoroquinolone and they inhibit DNA synthesis
- What is the basis of action of FQ’s? How do they act? They inhibit an enzyme that is central to DNA replication that initiates and causes negative super coiling to the DNA prior to splitting by helicase, called DNA gyrase aka topoisomerase. Both human and bacteria require DNA gyrase but there’s Type II topoisomerase that’s found in prokaryotes. The bacterial DNA gyrase (enzyme) differs from the eukaryotic DNA gyrase.
- The stupid way I remember fluoroquinolones inhibits TOPOISOMERASE activity… is that they are both big words with many syllables!
- The FQ is going to preferentially bind to the bacterial enzyme to a much lesser extent than the human gyrase.
- The major adverse effect of FQ is arthropathy (joint pain), primarily associated with a principal weight bearing joint because it inhibits the conversion of cartilage to bone. That is why children are most susceptible to this adverse effect but adults will get tendonitis, tendon rupture, bursitis as well.
- Another adverse effect is that it causes photosensitivity and photomutagenesis which are light-induced changes to DNA.
- FQ = Fluoroquinolones. ie.,
The product monograph is a quasilegal guideline document. As a patient you might get a package insert that gives you the adverse side effects and how you should take the dose and whatever else. First thing is shows is the warning. FQ’s are associated with an increased risk of tendinitis and tendon rupture (Is your ACL a tendon or ligament? It’s a ligament. Tendons are muscle to bone. What are ligaments? Bone to bone connective tissue. What’s the similarity? They’re both dense connective tissue).
- Pharmacology/Pharmakinetics tells you how the drug is distributed throughout the body which tells you the efficacy. The binding of cipro to serum proteins is 20-40% which isn’t likely to be high enough to cause significant protein binding interactions w/ other drugs.
What antibiotics inhibit Protein synthesis?
- Aminoglycosides (Proteins are made of amino acids, so aminoglycosides inhibit protein synthesis)
- Where have we seen gentamycin mentioned before?
- Gentamycin + Penicillin = synergistic effect against L forms.
- Gentamycin peak and trough = 10-12ug/ml and 1-2ug/ml
- peak is reached in 2 hours, trough in 4 hours. (symmetrical)
- side effects if too much: ototoxicity and nephrotoxicity
These antibiotics require monitoring. The therapeutic acceptable peak levels of these are 10-12ug/ml and the trough is 1-2ug/ml. The peak is usually 2 hours and the trough is 4 hours after administration. Peak and trough levels are a guide to the effective levels which are not to be exceeded to avoid toxicity. This is particularly important in patients with deficient kidney function. What would you do if levels exceeded peak and trough? You could lengthen the dosage frequency or lower the dose but you can’t arbitrarily do that because you have to consider the concentration achieved at the site of infection. If you need 5ug/ml in the CSF and you’re only getting 2ug/ml, is it going to be effective? NO. So you need to consider MIC, MBC and the concentration achieved at the site of infection.
Side effects: Ototoxicity and nephrotoxicity (middle ear damage and kidney damage) are the two primary consequences of aminoglycoside if peak and trough levels are exceeded, especially in elderly.
- Chloramphenicol (page 509, figure 21.14)
- Chloramphenicol is a BROAD SPECTRUM antibiotic! (review page 495 figure)
- In rare cases, chloramphenicol is fatal because it can cause aplastic anemia (the bone marrow completely stops producing blood cells). This happens in 1 out of 30,000 drug recipients and is inevitably fatal.
- Another threat is gray baby syndrome because newborns may die from multiple toxic complications so this drug is ONLY given to extremely ill hospitalized patients.
What antibiotics inhibit Folic Acid Synthesis? (page 502)
In this case we are looking at metabolism. FA is a precursor to the biosynthesis of nucleic acids. Trimethoprim blocks the pathway in which folic acid is converted into nucleic acids in bacteria but not in humans because humans use a different pathway to utilize that folic acid.
UTI: One of the most widely used antibiotics for treatment of UTI. Where do the bacteria primarily come from that cause UTI? The area between the genitalia and the rectum. Also, honeymoon cystitis is excessive sexual intercourse which predisposes to UTI.
Cranberry juice does not treat UTI’s, it reduces the incidence rate.
Pneumocystosis / Pneumocystic pneumonia is a form of pneumonia caused by a yeast-like fungus.
Sites of Action and Examples of Antibiotics Targeting EUKARYOTES (Fungi, Protozoa, Worms)
Folic acid synthesis
- trimethoprim (same as prokaryotes)
Nucleic acid synthesis
- flucytosine (RNA) an anti-fungal agent (coincidentally fluoroquinolones inhibit nucleic acid synthesis [DNA though] in prokaryotes, they both start with fl… hopefully that helps)
- amphotericin B
Antifungal drugs (pg 511)
- amphotericin b
- imidazole and triazole
Antiparasitic drugs (pg 512)
In Table 21.5: Remember Malarone for Malaria:
Malarone is the synergistic combination of 2 drugs: atovaquone and proguanil (he will probably ask this!)
Antiviral drugs (page 514)
- Acyclovir inhibits viral DNA
- Ribavirin inhibits viral RNA
Know how Acyclovir works!
Acyclovir is structurally similar to the nucleotide guanosine. An enzyme, found only in virus-infected cells converts acyclovir to acyclovir triphosphate, the actual antiviral drug. This false nucleotide competes with GTP to incorporate into DNA, leading to chain termination and stops DNA replication.
What is the major contraindication associated with antibiotic use? An indication is a circumstance of an infectious disease with characteristic etiology that warrants use of an antibiotic. Contraindications preclude to negate indications.
1. A history of allergic response or hypersensitivity is a contraindication, including anaphylactic shock which can be fatal.
2. Synergy: an amplified effect beyond an additive. The sum of the parts are more effective than their individual selves (1 plus 1 = 11). (malarone = atovaquone and proguanil synergistic combination)
Toxic effects of fluoroquinolones?
Arthrotopy, rupture of the ACL, tendinitis, bursitis, photosensitivity, photo-mutagenicity. The major problem is arthrotopy in every age group but particularly children. While you’re on fluoroquinolones, you have to avoid direct light.
ESBL = Extended spectrum beta-lactamase bacteria (beta lactamase 2.0)
With ESBL’s, if the organism is resistant to penicillin then it’s going to be resistance to beta lactam antibiotics because it means it has beta lactamase.
“The devilishly clever bacteria met our challenge by creating beta lactamase, an enzyme that grants many bacteria immunity to penicillin-type antibiotics. In turn, we upped the ante by developing new kinds of antibiotics that trounced these beta lactamase-producing pathogens.
But the bacteria weren’t done yet. Some tricky little bugs had a trick up their metaphorical sleeves: Beta Lactamase model 2.0, known to us as extended-spectrum beta lactamase, or ESBL. This enzyme not only chops apart penicillins, but cephalosporin antibiotics, too (all of the antibiotics whose generic names begin with “Cef-”).”
Page 278… Table 1.4, Know the problems associated with atypical bacteria.
The part about chlamydia.. they are atypical because they don’t have a cell wall … you can’t grow chlamydia on blood agar, it is an obligate intracellular pathogen so it must grow on tissue culture.
What are the 3 species of chlamydia that infect humans? Chlamydia pneumoniae, trachomatis (blindness, STD), and psittaci (causes ornithosis or psittacosis; parrot fever)
Chlamydia pneumonia. What is the primary significance? It causes pneumonia! Secondary significance: Coronary artery disease; myocardial infarction due to high antibody levels. People who have had heart attacks have high levels of antibodies which means they have been exposed to the bacteria.
Rickettsia: What is the only rickettsia that is able to survive outside the living host? Coxiella burnetii is the only rickettsia that could survive outside of the living host. It forms a spore like entity which isn’t equal to the bacterial endospore but can survive in soil, feces, etc for an extended period of time. It’s the only rickettsia that can do that and the etiology of Q fever.
Microscopy: Know the limits of resolution, the various types of microscopes (brightfield is the most common, electron microscopy is used for viruses, fluorescent is used for identifying antigens/antibodies and you don’t need isolation in pure culture to do fluorescent marking).
- Human eye: 200 micrometers.
- Brightfield: 200 nanometers (0.2 micrometer)
- Scanning: 10-20 nanometers
- Transmission = 1-2 nanometers.
You will be asked something like: How many times smaller of an object can be seen with a scanning electron microscope than the naked eye? Convert everything to nanometers to find out.
Review previous tests. Review those god damned presentation sheets. Review fucking everything. Good luck.
Mannitol Salt Agar
Mannitol provides the substrate for fermentation and makes the medium differential. The salt makes the medium selective because its concentration is high enough to dehydrate and kill most bacteria. Staphylococci thrive in the medium largely because of their adaptation to salty habitants, such as human skin. Most Staphylococci are able to grow on MSA but don’t ferment the mannitol so the growth appears with the unchanged pink or red medium color. S. aureus, however, ferments mannitol, which produces acid and lowers the pH of the medium. The result is bright yellow colonies. This test is specifically for isolating and differentiating S. Aureus.
- If there is no growth at all, the salt inhibited the organism, it’s not Staphylococcus.
- If there is good growth, the organism is not inhibited by the salt and it’s Staphylococcus.
- If there is good growth AND THERE’S A YELLOW HALO AROUND IT, then this organism is not inhibited AND it ferments mannitol, so it’s possibly S. Aureus!
- Moral of the story: If you see yellow, it’s probably S. Aureus.
Bile Esculin Test
In this medium there is bile and esculin. Bile is the selective agent used to separate Streptococcus bovis and enterococci from other streptococci. Esculin is present because only the group D streptococci and enterococci can hydrolyze esculin in the presence of bile salts.
- Any blackening of the medium means Bile-Esculin positive and presumptive identification of group D streptococcus or Enterococcus.
- Note, if there is blackening of the medium before 48 hours, it can be recorded as Bile-Esculin positive.
- If there is no darkening after 48 hours, it is not a group D streptococcus or Enterococcus and is Bile-Esculin negative.
- Incubation for at least 48 hours must pass for the negative result to be determined.
This is a selective and differential medium for the growth of gram-negative bacteria and contains lactose and it’s dark brown colored. If the bacteria on it can ferment the lactose, the acidity turns the dye into a bright pink color.
- If there is poor or no growth, the organism is inhibited and it’s probably gram-positive.
- If there is good growth, then it’s gram-negative.
- If there is good growth AND it’s red, this means the gram-negative bacteria is producing acid from lactose fermentation and it’s probably a coliform like E. coli.
- If there is good growth but it’s not red, then it’s a noncoliform bacteria.
Eosin Methylene Blue Agar (EMB Agar)
This is a complex, selective and differential medium used to isolate fecal coliforms. The dyes inhibit the growth of gram-positive organisms (like the MacConkey) but they also react with lactose fermenters, turning the growth dark purple or black. If it’s a black growth with green metallic sheen, it’s likely E. coli. If it’s a pink color, it’s a less vigorous lactose fermenter. If there is a lack of color, it’s a nonfermenter.
- If there is poor or no growth, the organism is inhibited and it’s probably gram-positive (like the MacConkey)
- If there is good growth, then it’s gram-negative (like the MacConkey)
- If there is good growth and it’s “dark” purple or black, it’s probably a coliform like E. coli.
- If there is good growth AND it’s red/pink, it’s probably a coliform like Enterobacter or Klebsiella.
- If there is good growth but it’s not pink, purple or metallic and there’s no color, it’s a noncoliform bacteria (because it doesn’t ferment lactose or sucrose).
Normally this broth looks clear/straw-colored and the top is slightly pink due to oxidation of the dye with its contact to oxygen. This medium can be used to grow microbes representing all levels of OXYGEN TOLERANCE but it’s generally associated with cultivating strict anaerobes and microaerophiles. A microaerophile requires oxygen to survive, but environments containing lower levels of oxygen than the atmosphere.
So let’s go over what these results mean:
- If only the top has growth, that means it is a strict aerobe because it is growing only in the top part.
- If the bottom has growth, it means it’s a strict anaerobe (aka obligate anaerobe) because it can grow in the portion furthest away from the oxygen.
- If there is growth throughout, but mostly at the top, it is a facultative bacteria since it likes aerobic respiration best but can grow without it as well.
- If there is only growth just below the very top, that’s indicative of a microaerophile, because it needs oxygen to live, but not a lot of oxygen, so it’s not going to grow closest to the top.
- If there is growth throughout evenly diffused, that’s indicative of an aerotolerant anaerobe as the presence of oxygen doesn’t affect them.
Oxidation-Fermentation Test (O-F)
This test is designed to differentiate bacteria on the basis of fermentative or oxidative metabolism of carbohydrates. In fermentative pathways, when the carbohydrate is broken up, there is acid production present. Two tubes are used, one with mineral oil to promote anaerobic growth/fermentation and the other has no mineral oil to allow aerobic growth and oxidation.
- The control is green. If both tubes turn yellow, it means the organism can ferment the carb AND oxidize the carb in both scenarios.
- The control is green. If both tubes remain green, there is no sugar metabolism and the organism is nonsaccharolytic.
- If the sealed tube remains green, but the unsealed becomes yellow, it has oxidative fermentation.
- If there is slight yellowing only at the top of both tubes, it’s oxidative and/or slow fermentation.
- Note: A fermentative organism (F) will look exactly the same as an organism capable of both oxidation and fermentation (O-F).
- Oxidation = Pseudomonas and Alcaligenes
- Fermentation = E. Coli (facultative)
So it’s very easy, in a O-F tube, it starts as green, any yellowing indicates the breakdown of carbs and whether it has mineral oil or not determines under what conditions the bacteria can do it under.
Phenol Red Broth
This is a broth to which a carb such as lactose or sucrose is added and there is a durham tube that is used to indicate gas production.
Methyl Red and Voges Proskauer Tests
The MR test is used to detect organisms capable of performing a mixed acid fermentation.
MR and VP are the same media to start with.
- MR: E. Coli is positive. E. aerogenes is negative.
- VP: E. coli is negative. E. aerogenes is positive.
- Catalase positive = Staphylococcus epidermidis
- Catalase negative = Enterococcus faecalis
- Gas presence; nitrate positive = E. coli
Used to determine if the organism has the ability to use citrate as its sole source of carbon. Part of the IMViC (Indole, Methyl Red, VP and Citrate) tests to differentiate members of Enterobacteriacea and other gram-negative rods.
- Positive if blue = Enterobacter aerogenes
- Negative if green = E. coli
Just like the citrate test, it’s used to differentiate organisms in the Enterobacteriaceae family.
- Purple is the only positive result and indicates that the organism produces the decarboxylase enzyme.
- Enterobacter aerogenes is positive.
- All other results are negative.
Phenylalanine Deaminase Test
Organisms that produce phenylalanine deaminase can remove the amine group (NH2) from the amino acid phenylalanine. The phenylalanine agar is used to differentiate the genera Morganella, Proteus and Providencia from other members of the Enterobacteriaceae.
The agar is normally yellow. If the phenylalanine deaminase is present, then when phenylalanine is broken down, there will be phenylpyruvic acid present and when you add ferric chloride, it will turn green.
- A dull military green color is a positive result indicating the presence of phenylalanine deaminase.
- Proteus vulgaris is PD positive.
- No color change means PD is absent.
- E. coli is PD negative.
Urea is a product of decarboxylation of certain amino acids. It can be hydrolyzed to ammonia and CO2 by bacteria containing the urease enzyme. Urea agar was formulated to differentiate rapid urease-positive bacteria from slower urease-positive bacteria and urease-negative bacteria.
Control looks orange.
- If it turns ALL pink within 24 hours, that indicates rapid urea hydrolysis; strong urease production.
- If it turns partially pink within 24 hours (or all pink after 24hrs), that indicates slow urea hydrolysis.
- If it remains orange within 24 hours and then all or partially pink after 24hrs, that is also slow urea hydrolysis.
- If it remains orange or yellow, no urea hydrolysis; urease is absent.
- E. coli is urease negative.
- Proteus vulgaris and Klebsiella pneumoniae are urease positive.
Gelatin Hydrolysis Test
This test is used to determine the ability of a microbe to produce gelatinases. If the gelatin turns to liquid, gelatinase is present.
- S. Aureus is gelatinase-positive and can be differentiated from S. epidermidis.
- Serratia and Proteus are gelatinase-positive and can be differentiated from other enterobacteriaceae.
- Bacillus anthracis, B. cereus are gelatinase positive, as are clostridium tetani and c. perfringens.
- E. coli is gelatinase negative.
Triple Sugar Iron Agar / Kligler Iron Agar
You have to know how to read slants and butts. You typically read the slant first, then the butt.
- A yellow color indicates fermentation with acid accumulation
- A pink/red color indicates protein catabolism with alkaline products.
- Black precipitate indicates Hydrogen Sulfide (H2S).
- A/A+ = Acid (yellow) slant / Acid (yellow) butt
- K/A = Alkaline (red) slant / Acid (yellow) butt
- K/K = Alkaline (red) slant / Alkaline (red) butt = Not enterobacteriaceae
- K/NC = Alkaline (red) slant / No change in butt = Not enterobacteriaceae
- NC/NC = Organism isn’t growing at all = Not enterobacteriaceae
- H2S = Black precipitate indicates sulfur reduction.
Summary of what we know about E. coli
- E. coli grows pink/red in MacConkey agar
- Grows dark purple with a green metallic sheen on the EMB agar,
- it’s fermentative (facultative) in the O-F test,
- positive in the Methyl Red test,
- negative in the VP test,
- nitrate positive,
- citrate negative,
- phenylalanine deaminase negative,
- urease negative,
- gelatinase negative
1. Identify the bacterial organelle shown. What is its function? Give the gram reaction.
- Organelle: Capsule.
- Function: Cytotoxic.
- Gram reaction: Indeterminate because it’s not a gram stain.
2. Name the plant pathogen shown. What is its significance?
- Pathogen: Claviceps purpurea.
- Significance? Potent hallucinogen used in religious ceremonies / Causes ergotism / Predisposes to gas gangrene (pick one of these).
3. Liver biopsy of a marine recently returned from Afghanistan. Identify the organism genus/species, disease and vector.
- Organism. Leishmania Donovanii.
- Disease. Leishmaniasis / Dum dum fever / kala-azar
- Vector: Brown Sand Fly Vector
4. Monogamous female with a week long history of a foul smelling creamy discharge. Identity? Give the stage. How is it acquired?
- Identity: Trichomonas Vaginalis
- Stage: Trophozoite; No cyst stage, also motile due to presence of flagella.
- Acquisition: Sexual Transmission
5. Blood smear of a native from Sudan with a 3 week history of recurring fever. Give the organism genus/species and vector.
- Plasmodium malariae
- Disease: Malaria
- Vector: Female anopheles mosquito.
6. Name the parasitic flatworm shown. Give the common name and where in the world is this expected to be found.
- Identity: Clonorchis Sinensis.
- Where is this found? Asia (EE’s backyard)
- Common name: Asian Liver Fluke
7. Name the Structure shown, name the organism from where this is taken and how is it typically acquired?
- Scolex structure.
- Organism: Taenia solium.
- From undercooked pork/wild game
8. Name the colonargea shown. What stage is it? What is its significance?
- Colonargea volvox.
- Stage: Zygospore.
- Significance? Important evolutionary basis for multicellularity.
9. Identify the common fungus shown. Give the stage. Significance?
- Fungus: Rhizopus spore
- Stage: Zygospore stage.
- Significance: Opportunistic mycosis in compromised host.
10. Name the common environmental fungus shown. What is its significance?
- Fungus: Aspergillus.
- Significance: Causes lung/ear infections (black ear disease; very common in farmers) / Aphylatoxin is produced. opportunistic mycosis in compromised host.
11. Found in normal flora in gut.
- Candida albicans.
- Fungal vaginitis (yeast infection); diaper rash; esaphagitis in AIDS patients.
12. Name the fungus shown, what is its significance?
- Rarely associated with infection.
13. Name the parasitic flatworm, give the common name, how is it acquired?
- Genus/species: Fasciola hepatica
- Common name: Sheep liver fluke
- Acquired by contaminated game.
14. Blood smear of a native from wherever. Name the organism, vector and disease.
- Trypanosoma organism
- African sleeping sickness
- Vector: tse-tse fly.
15. Gram stain of a 25 year old promiscuous male who thinks only wimps wear condoms.
- Neisseria Gonorrhea.
- Diasese: Gonorrhea / The Clap / French toilet
- Transmission: STD
16. Name the bacterial organelle show. Give the function. Suggest the identity based on the characteristic morphology.
- Organelle: Flagella
- Function: Motility
- Organism: Proteus Vulgaris
17. Trichrome stain of a volunteer who returned from Ecuador with bloody diarrhea. Identify the organism, give the stage, associated disease and how is it acquired.
- Organism: Entamoeba Histolytica
- Stage: Trophozoite
- Disease: Amebiasis
- Acquisition: Fecal contaminated water.
18. Muscle biopsy from a football player fed smoked mountain lion by his coach. Name the organism, disease and how is this acquired.
- Organism: Trichinella Spiralis
- Disease: Trichinosis
- Acquired: Contaminated/undercooked meat/game
19. Trichrome stain of a 2 year child who is not potty trained with foul smelling, explosive diarrhea. Give the organism, stage, disease and acquisition.
- Organism: Giardia lamblia
- Stage: Cyst because it has four nuclei
- Disease: Giardiasis (Beaver fever)
- Acquisition: fecal contaminated water.
20. Name the bacterial organelle shown taken from a facultative bacteria. Give the gram reaction and how do you know this.
- Organelle: Endospore.
- Gram reaction: Positive because only gram positive bacteria form spores.
- Likely identity: Bacillus
21. Identify the bacterial structure. Suggest the likely identity since this is from an obligate anaerobe.
- Organelle: Endospore.
- Gram reaction: Positive
- Organism: Clorstridium perfringens.
22. A gram positive, obligate anaerobic organism was isolated from the necrotic tissue of this truck driver. Identify the organism, diagnose the disease.
- Organism: C. perfringens.
- Disease: Gas gangrene
23. Fingers and back of a hairdresser 2 days after surgery from a broken hip. A gram positive, coagulase positive, catalase positive organism was isolated from the blood. Identify the organism, diagnose the disease, what’s unusual if it is?
- Organism: S. aureus.
- Disease: Post operative toxic shock.
- This is common in women and unusual in men.
23. Trichrome stain from 2 year old with explosive diarrhea. (compare this with #19!)
- Organism: Giardia lamblia.
- Stage: Trophozoite
- Disease: Giardiasis (Beaver fever)
- Acquisition: Fecal contaminated water
24. Name the pattern hemolysis show and one organism given this pattern.
- Hemolysis: Beta.
- Organisms: C. perfringens / S. aureus. / S. agalactiae / Strep pyogenes.
Every material has a property known as heat conductivity that tells you how good that material is at conducting heat. When you touch an object, it will remove heat from your hand at a rate proportional to its conductivity. In other words, it’s all about the rate at which it can conduct heat.
Carpet is mostly air and air is a poor conductor of heat. Wood is solid, so it is a better conductor. Tile and metal are even better conductors than wood. When you touch the carpet, it is not able to remove heat from your feet as easily as wood, and as a result, it feels warmer. Conversely, wood or tile or metal is able to remove heat from your foot very easily, and so it feels colder. Now you know why sitting on that porcelain toilet seat in the morning feels cold and why wearing socks works so well at insulating your feet.
It’s probably worth mentioning that this coolness depends on your foot being warmer than the surface it is touching. If the floor was hotter than your feet, tile would feel hotter than carpet because it could transfer heat to your body faster. This is why a backyard that is made of stone that has had the sun beating down on it all day can feel super hot and you might quickly jump back onto the door mat or in the shade to get some relief.
Note that this doesn’t have anything to do with specific heat, which is something else entirely. Specific heat has to do with how much energy is needed to raise an object’s temperature. Heat conductivity is all about the rate at which the material can conduct heat. Simply put, tile is the better conductor of heat than wood or carpet and can take away the heat from your feet faster than the others.
Obtaining a pure culture is mandatory to establish the identity and susceptibility. Everybody knows what a pure culture is, right? It’s a culture consisting of a single type of microorganism derived from a single cell. When the specimen arrives in the lab, you have to isolate the bacterias, then take your inoculum and you put it on a sterile. If there’s multiple bacteria, like e.coli and staphylococcus, you have to get separate cultures of each before doing a pure culture. Only then can you do the C&S report.
Let’s say someone goes to the doctor because they have a sore throat. The doctor takes a specimen. There’s several types of bacteria it could be (E. coli, S. aureus, pseudomonas, etc). For it to be identified, you can’t do the test with all three present. The bacteria have characteristics that are unique to themselves. So we give them what they want (food) to make them happy. We put them in selective media to do that.
The Mannitol Salt Agar (MSA) favors the growth of S. aureus because the salt inhibits most bacteria. Remember S. aureus is salt tolerant and grows up to 10% salt concentrations but minimally at 15%. It then ferments mannitol salt which ferments large yellow colonies, so you could ensure that’s s. aureus. So that selective media works only with specific types of bacteria.
The Eosin Methylene Blue (EMB) favors the growth of fecal coliforms such as E. coli. In EMB it becomes a blue/black color.
The MacConkey agar is used for growing gram negative bacteria because it inhibits gram positive bacteria. In MacConkey’s, the agar changes to a pink color. If you ever mix gram + and gram – and you put that mix onto MacConkey agar, which bacteria are you going to get growing? Only the gram negative version.
Candle jars must be provided for microaerophiles. Microaerophilic bacteria require oxygen for growth but at lower concentration than is present in the atmosphere.
The point of this selective media is that the bacteria don’t grow if the medium and incubation conditions are inappropriate.
Side note: When you have an organism that’s difficult to grow, you use a DNA probe test. For example, chlamydia is very difficult to grow, so they will just use DNA. In fact in UCLA and other labs, the screening for gonorrhea is based on genomics. You can’t do a pure culture when you have a 1,000 people coming in to a hospital everyday.
Sterilization usually involves autoclaving which involves heating (121°C/250°F) under high pressure (15psi) in an autoclave. An autoclave is like a big pressure cooker. This allows things to reach higher temperatures than otherwise would be possible because it forces a higher vapor pressure to build up so water will boil at a higher temperature to kill things (conversely, water boils at lower temperatures at high elevations where the air pressure, aka the vapor pressure, is lower. If you’re camping up at 12k ft elevation, you can’t even get the water to be hot enough to cook food when it boils because it will boil at 70 degrees! ::the more you know star across the screen goes here::).
You can’t use autoclaving on heat sensitive materials such as proteins. You therefore use filtration to sterilize them. There are other methods which we’ll look at but for our purposes right now, the most common process is the autoclave.
The most common procedure used for pure culture isolation is the quadrant streak isolation. When you have cross contamination and multiple appearing colonies, that’s not a pure culture.
You need to get a loop of the specimen, streak it on one section, flame and cool, turn it 45 degrees and streak a second time, overlapping the first streak. Then repeat it a third and fourth time. If done properly, you’re going to get isolated regions in the third region typically and if there were three different organisms, you’ll get separate colonies or all three. Then you could take each of these to a separate plate and then you do a streak and then you get pure culture on a separate plate.
Why does the flaming and cooling separate them? It dilutes the cultures over the plate. If you don’t flame/cool you’ll never get isolation. You have the organisms together and you separate them, so you spread them out.
Pour plate and spread plate methods are not used for isolation and pure culture clinically.
Other procedures are tedious and counter productive. When you do the pour plate, it’s used for literally counting the bacteria in a clinical specimen. That would be impossible because you need a gigantic room just to do one organism. You would put it on different media and multiply the number of bacteria on the plate, by the dilution. The only results that are countable are the ones where you could count the dilution with 30-300 CFU/mL. If you have to do this for every single specimen, it would be counterproductive because you MAY get isolated colonies. The pour plate and the spread plate are more used to count the number of specimen in juice, water, milk, anything, etc. The only time you use that to diagnose for a clinical reason is for UTI. How is UTI defined? The isolation of a >100,000 (10^5) CFU/mL. We learned this during the case history chart analysis.
Viability stains are contradictory because the staining itself kills all the organisms, but it’s used to tell which one was dead from the get go and which one was alive. There’s two types of viability stains, Methylene Blue and Trypan Blue Exclusion.
Methylene Blue is used for yeast viability in beer, wine, alcohol. Viable (living) yeast do not stain and remain colorless; whereas non-viable, non-metabolizing cells stain blue.
Trypan blue is used on animal cells and the results are the same as MB. Since cells are very selective in the compounds that pass through the membrane, in a viable cell, trypan blue is not absorbed, or in other words, it is excluded, which is why it is sometimes called a dye exclusion method. In dead cells though, the dye passes through and appears blue under a microscope.
Critical Values in Microbiology are results that indicate a potentially life threatening condition requiring immediate attention by the clinician. Critical Laboratory results are always reported by telephone immediately. If any of these come positive, they will be reported to the physician…
Positive Blood Culture initially detects organism by gram stain.
Positive CSF Gram Stain or Culture
Positive Directigen. Directigen is an antigen, meaning it’s part of the organism, and is a characteristic of it. You’re using this as a method of identification. You’re looking for the antigen basically and it’s generally done in blood or blood products.
Positive AFB Smear or Culture indicates tuberculosis or leprosy.
Positive India Ink typically means a negative stain which is done for fungi and is typically associated with meningitis so the specimen of choice is CSF and that identification is done by a Negative stain.
Positive C. difficile Toxin indicates a diagnosis of Pseudomembranous colitis, or as it’s more commonly called, antibiotic associated colitis
Uses / Clinical Applications
1. The gram stain is the single most useful staining procedure for the rapid presumptive ID of bacteria clinically as the basis of the preliminary report for the C & S.
2. Suggests possible identify based on gram reaction, morphology and arrangement. Gram negative diplococci (neisseria). Gram positive cocci (staphylococcus). Gram negative rod (e.coli, psuedomonas). You can’t tell e. coli apart from pseudomonas but it may not be necessary to refer treatment because certain antibiotics are known to be effective against gram negative bacteria.
3. If a gram stain is ordered STAT it could take 30-40 minutes as opposed to 12-24 hours.
4. Provides preliminary data on susceptibility to antibiotics for treatment. If the bacteria are still there after antibiotic was given, then it didn’t work.
5. Suggests media, incubation conditions for growth and isolation of bacteria from clinical specimens. For example, if you had a bite wound that suggested anaerobic bacteria, would you have to tell the lab you suspect anaerobic bacteria? Yes. Because if you set it up aerobically, the results will be meaningless. Clinical labs usually have standard operating procedures to eliminate these issues and maximize recovery. When you take a blood culture you take two cultures, one anaerobic and one aerobic.
6. May explain failure to obtain viable organisms on culture possible due to inadequate growth media and or incubation conditions: Anaerobes incubated aerobically.
Advantages: rapid, cost effective, potentially life saving.
The gram stain procedure
Gram positive bacteria have a lot of peptidoglycan (which is part of the bacterial cell) and a tight/dense matrix to retain the primary stain. If you look on page 85 of your text book, it shows you the 3 types of cell walls. The crystal violet is going to be retained by the thick dense matrix within the peptidoglycan. When you add the alcohol, the dye remains because it’s bound firmly to the cell wall because of the thick matrix. Imagine you have a piece of net, imagine it is just one layer vesus several layers of net. Imagine you pour sand through the multiple-net layer, the sand will stay because of all the peptidoglycan. If the matrix is loose, the primary dye is not retained. You add iodine which increases the affinity for the dye to attract to the cell wall.
When you add the alcohol, the primary crystal violet dye is washed out in a gram negative bacteria but remains in the gram positive bacteria. At this stage the gram positive bacteria will be purple and the gram negative will be colorless.
When you add safranin (red dye) what happens to the gram positive? It will remain purple but the gram negative will become red. So you end up with gram negative that is pink/red or gram positive that is purple. (This is shown on page 59.)
Role of gram stain in C & S
The gram stain is used as the basis for treatment and is potentially life saving in critically ill patients with meningitis or sepsis. A clinician cannot wait for the complete work up of a C & S (3-5 days) to treat someone in septic shock. The gram stain could be done in 30 minutes stat or typically within 2-4 hours. The gram stain is part of the critical values data set and it’s called critical because it’s pivotal/essential/potentially-life-saving.
Role of acid fast stain in C & S
Relatively few bacteria are acid fast bacteria but it can be useful to form basis for tuberculosis or leprosy. The two genera that are AFB are actinomycetes and mycobacterium. By far the most common genus is mycobacteria and the most common bacteria are M. tuberculosis and the other is M. leprae. There are others such as M. Avium intracellulare and M. chelonae and yadda yadda yadda.
An AFB stains red, but it is a different procedure than a gram stain so it has nothing to do with that red. AFB have gram positive cell walls so they stain red (and not purple) because it’s a different procedure. You wouldn’t do a gram stain on a AFB (like if you suspect someone has a tuberculosis) because if you did the gram stain the bacteria would be gram positive and wouldn’t tell you it’s tuberculosis (or a AFB). The acid fast stain provides a more definitive diagnosis since it targets all mycobacteria. The presence of one single AFB in sputum or tissue confirms a diagnosis in most cases of tuberculosis.