DONT TRUST MY SPELLING!!!
- • Selective – only grows certain types of organisms. Ex. EMB selects for gram – organisms and inhibits the growth of gram +.
- • Differential – what organisms do when they grow. Ex. EMB is differential based on lactose fermentation
- • Enriched media – don’t know exactly what is in it, really good for fastidious organisms (organisms that require a lot of ingredients)
- • Chemically defined – all components are known. Organisms that grow on these are the complete opposite of fastidious. Ex. M-9, E. coli grows on this
- • General purpose – use this if you want to grow an organism and don’t need anything special to do it
- • NA Plates – general purpose. We used to isolate organisms from our body and from dust.
- • TSA (triptic soy agar – has soybean digest and triptone which is a digestive kasin. Kasin is the protein that makes milk white)– general purpose. We used it to isolate organisms from our body and from dust.
- • Nutrient Agar – has beef extract and peptone for nutrients.
- • Motility test agar stab tube – differential, used to determine motility. Percent of agar used is 0.5%. The plates have 1.5% agar.
- • Sab-Dex Plates – selective because they only grow fungi. High sugar and low pH around 6 which is great for fungi growth.
- • Agar block plates – made with sab-dex. Used to identify different molds.
- • Blood agar – enriched media. Differential because you can look at hemolysis. Alpha hemolysis – partial clearing of red blood cells. Two organisms that did that are streptimoni and strep salivarius. Beta hemolysis – complete clearing. Two organisms that do this are streptococcus pyogenes and staph aerues.
- o How do you make blood agar/chocolate agar → start with a base (TSA,NA, Brain Heart infusion). Heat it and melt it. Cool it down to 35-40C before you add 5% sheep blood.
- o Chocalate Agar is the same process except you cool it to 80C when you add the blood. This lyses the blood cells and turns it a chocalatey color.
- • Chocalate Agar – enriched media. You can grow even more fastidious organisms on it. It is the same principle as cooking food → it helps the organism digest nutrients easier. You would grow strep pnemonea, any legionella, heamophilus influenza, neisseria gonohrrea.
- • EMB plates – selective and differential. Selective because of the dyes (eosin and methaline blue → inhibit gram (+) this means they select for gram (-)). They are differential because of lactose.
- o EMB – e. coli has metallic green sheen
- o Klebsiella – gooey fish eye
- o EA – looked normal
- o Proteus Mirabilis – negative
- o Staph Aureus – had trouble growing
- o Ent. Feacalis – had trouble growing
- o B. sub – had trouble growing but had endospores and survived.
- • MSA plates – Mannitol Salt Agar. Mannitol makes it differential. Salt makes it selective. It differentiates based on mannitol fermentation. Also has pH indicator → phenol red. In the presence of an acid it turns yellow. Same pH indicator used in TSI slants and Urea agar slants. When it is basic turns red. A positive reaction on MSA is a yellowing of the agar, negative is still pink.
- o It selects especially well for staphs. They can tolerate the high salt level. You can also grow entrococcus on it → positive but did not ferment as well as staph aeurues.
- • M-ENT plates – extremely selective, only grow enterococci. The ingredient that makes this so selective is sodium azide. Similar to cyanide. It also has a pH indicator TTC → doesn’t make it differential just makes the colonies turn a red color when the grow making it more visible.
- • BHI agar – enriched medium. Good for fastidious organisms. Can make blood/chocolate agar with it.
- • Starch agar plates (NA plate with starch in it) – differential. We used them to differentiate organisms ability to break down starch. B. sub could break down starch. E. coli could not break down starch. We are ultimately testing for the presence of amylase → this breaks down starch.
- • TSI slants – highly differential. Three sugars, has iron in it. Three sugars are lactose (10x), sucrose (10x), glucose (1x). Also has iron sulfate which helps with H2S production in some organisms. Has pH indicator Phenol red (yellow with acid, red with bases).
- • 3 things to look for sugar fermentation, gas bubble production, H2S production.
- • Only proteus do iron to H2S
- • Skim Milk Agar plate – differential. NA plate with skim milk to give them some kasin. Testing for presence of a protease or kasinase.
- o B sub – positive (has protease)
- o E. coli
- • Nutrient gelatin stab tubes – differential. Gelatin is a protein matrix – that’s what keeps it solid. If an organism has a protease it will break down that protein matrix. A positive reaction would be a liquid tube. Negative reaction – tube still solid. Used it looking at mostly gram (-)
- • Plate Count Agar – like NA, general purpose
- • Simmons Citrate Agar Slant Tubes – differential. Used for Gram (-) rods. PH indicator is Brohm Thymol. Starts out green – in a presence of a base it turns blue. We are testing for the ability of an organism to utilize citrate as a source of carbon. If they can they will grow. Also present are ammonium salts → if it grows it will use these salts (create ammonia). The ammonia then reacts with the Brohm Thymol and turn it from green to blue (blue is the positive reaction for this one). Used for gram (-)
- • Urea Agar Slant Tubes – used for gram (-). Differential. Testing fro presence of urease. Urea broken down to CO2 and ammonia using a urease. The pH indicator is phenol red. In presence of a base it goes pink. Positive is pink.
- • Mckonkey Agar Plate- just like EMB but with different ingredients. Used for gram (–) organisms. Unlike emb it selects for a more narrow range of organisms. Two of its ingredients make it selective – bio-salts and crystal violet (inhibit gram (+) org). The lactose makes it differential. It has a pH indicator – neutral red. When in the presence of acid it turns pink. Positive reaction is pink meaning lactose fermentation. Negative is clear/see through.
- • MH - Muelerr Hinten Agar – tests for antimicrobial sensitivity. Has beef extract. General purpose.
- • Enterotubes – differential. Results only meaningful if using gram (-) rods.
- • Liquid broth
- • Litmus milk – differential. grow tons of stuff. Looking for three things: lactose fermentation, curd formation (soft or hard), proteolisis (zone of clearing of kasin). Two ingredients = litmus and skim milk.
- • Tryptone broth – used to detect presence of tryptophanase. Tryptone digest of kasin is rich in tryptophan. If an organism makes tryptophanase it breaks it down and results in indole. Put kovacs reagent in and it reacts with indole. If it is positive it will produce a red ring on top (negative- no reaction). Used a lot for gram (-).
- • Nitrate broth – gram (-). It is differential. It is basically beef extract but rich in NO3. NO3→NO2→N2. Positive reaction is the gas bubble. Only one organism was able to make it through and do this → Pseudomonas Auriginosa
- • Rabbit plasma (not as important) – differential. Staph aerues is (+). This means it produces coagulase
- • 35min
- • Lactose broth – differential. Used for water microbiology. Positive reaction is an air bubble. The air bubble in lactose broth comes from lactose fermentation. When you ferment lactose you make acid and a gas byproduct. This test is used to test for gram (-) rods because they will do this.
- • MR-VP broth – what organisms do with glucose. Glucose could be fermented to organic acid which lowered the pH to 5ish. Methyl red is added after incubation - If glucose fermented red appeared in tube. OR . . . VP - glucose is converted into 2,3 butane diole. Barets reagent added to convert 2,3 butane diole into acetoine which would give tube a red color. Good test differentiating gram (-) rods.
- o e. coli → positive for methyl red. Turned glucose into various natural acids
- o e. a. → negative for methyl red
- o Using the OTHER method
- o E.a → positive. Made 2,3butane from glucose
- o E.c. → negative
- • M-9 – chemically defined. E. coli grows on it.
Things to know
- • Gram reaction, shape
- • Do they ferment lactose, do they make spores, do they make pigment
- Ex. Psuedomonas aureiginosa – makes extracellular pyocyanin pigment
- Serratia marcesins – makes intracellular prodigiosin
- ML – makes pigment
- Pseudomonas flourescens – makes pigment
- • Know if they are bacteria, yeast or mold, ect. And any notable reactions.
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