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What are the 3 chemical reagents that are used in differential staining?
1. Primary stain
2. decolorizing agent
What does Gram staining do?
it divides bacterial cells into two major groups - gram-positive and gram-negative
What is the gram stain reaction based on?
The difference in the chemical composition of bacterial cell walls
What kind of cell wall does Gram-negative cells have?
thinner and surrounded by outer lipid-containing layers
What does the iodine accomplish?
increases the cells' affinity for a stain and does this by forming an insoluble complex which intensifies the stain
What does the decolorizer do?
in gram negative cells the alcohol increases the porosity of the cell wall by dissolving the lipids in the outer layers
What are the steps to prepare a slide for staining? (4)
1. clean slide
2. label slide on front and back
3. thin smear
4. heat fix
What does selective media do?
selective media is used to select (isolate) specific groups of bacteria. The inhibit the growth of one type of bacteria while permitting growth of another, thus facilitating bacterial isolation.
What are the 3 types of selective media?
1. Phenylethyl alcohol agar
2. Crystal violet agar
3. 7.5% sodium chloride agar
What is phenylethyl alcohol agar used for?
the isolation of most gram-positive organisms - it inhibits the growth of gram negative organisms
What is crystal violet agar used for?
is selective for most gram-negative microorganisms - inhibits gram-positive
What is 7.5% sodium chloride agar used for?
inhibitory to most organisms other than halophilic - most useful in the detection of members of the genus Staphylococcus
What does differential/selective media good for?
They incorporate chemical compounds that following inoculation and incubation produce a characteristic change in the appearance of bacterial growth and/or the medium surrounding the colonies which permits differentiation
What are the 3 types of differential/selective media?
1. Mannitol salt agar
2. MacConkey agar
3. Eosin-methylene blue agar
How does Mannitol salt agar work?
this medium contains a high salt concentration which is inhibitory to the growth of most but not all bacteria other than the staphylococci it ALSO contains the carbohydrate mannitol which can be fermented by some staph - it produces a yellow zone surrounding their growth
How does the MacConkey agars work?
1. uses crystal violet to inhibit growth of gram-positive organisms
2. uses lactose fermentation
3. coliform bacilli - produce acid as a result of lactose fermentation - "red colonies" (E. coli)
4. Dysentery, typhoid and parathyphoid bacilli are not lactose fermenters and do not produce acid - they appear tan and frequently transparent
How does Eosin-methylene blue agar work?
lactose and the dyes eosin and methylene blue permit differentiation between enteric lactose fermenters and non-fermenters as well as id E. coli (which would appear blue-black with a metallic green sheen)
*gram-negative growth is more abundant
What is enriched media? examples.
supplemented with highly nutritious materials such as blood, serum or yeast for the purpose of cultivating fastidious organisms; extract - like blood agar
i.e. blood agar, chocolate agar, mueller-hinton agar
What hemolytic results can you see from enriched media?
1. Gamma hemolysis - no lysis
2. alpha hemolysis - incomplete lysis (greenish halo around the bacterial growth)
3. beta hemolysis - complete lysis (has clear zone around the bacterial growth)
What does the triple sugar-iron (TSI) agar test for?
differentiate amoung the different groups of enteric bacteria; differentiation is made on the basis of differneces in carbohydrate fermentation patterns and hydrogen sulfide production
TSI - What does a result of a red slant and yellow butt with or without gas production mean?
alkaline slant; acid butt; only glucose fermentation has occurred; bacteria likes glucose first
TSI - What does a result of a yellow slant and yellow butt with or without gas production mean?
acid slant; acid butt; lactos and/or sucrose fermentation has occurred
TSI - What does a result of a red slant and a orange/red butt mean?
No carbohydrate fermentation has occurred
What are the 3 types of enterics found in the intestinal tract of humans?
2. occasional pathogens
3. normal intestinal flora
What 3 types IMViC tests can be done to identify enteric bacteria.
2. methyl red
3. citrate utilization
(there is Voges-Proskauer also)
What does indole test for and what results can you get?
1. indole test for the bacterias ability to hydrolyze tryptophan with the production of indole; indole is detectable by adding Kovac's reagent.
2. Cultures producing a red reagent layer after adding Kovac's reagent are indole-positive
Absence of red coloration is indole-negative reaction
3. examples: E. coli, P. vulgaris and E aerogenes
What does methyl red test for and what results can you get?
1. test for what enzymatic pathways bacteria uses glucose and what the end products are.
2. Great for separation of E. coli and E. aerogenes
3. E. coli - has low pH = red color
E. aerogenes - has a higher pH = yellow color
What does citrate utilization test for and what results can you get?
1. without glucose or lactose, some bacteria can use citrate as a carbon source for energy.
2. citrate-positive= growth on surface and blue coloration
citrate negative= no growth and slant stays green
3. examples: E. coli, E. aerogenes, and K pneumoniae
What does SIM agar test for and what results can you get?
identify enteric pathogens
1. looking for hydrogen sulfide production. A black color indicates H2S production
2. Diffuse growth from the stab line or cloudiness of the medium indicates motility
3. 1-2 drops of Kovac's reagent - red to a dark cherry red layer - is indole positive
What does Urease test for and what result can you get?
1. urease is a hydrolytic enzyme that attacks the nitrogen and carbon bond in amide compound such as urea and form the alkaline product ammonia.
2. ammonia makes test positive and turns yellow slant (or broth) bright pink
no pink=no reaction
3. examples: Klebsiella pneumoniae, Proteus vulgaris
What does Catalase test for and what result can you get?
1. common enzyme found in nearly all living organisms exposed to oxygen. It catalyzes the decomposition of hydrogen peroxide to water and oxygen.
2. adding the substrate hydrogen peroxide to TSA culture; if it bubbles - it tests positive
no bubbles - negative results
What does Phenylalanine Deaminase Test for and what result can you get?
1. if organism possesses phenylalanine deaminase, phenylpyruvic acid will be released into the medium and can be detected by the addition of a 10 to 12% ferric chloride solution to the surface of the medium.
2. green color develops upon the addition of ferric chloride it is positive
What ways control microbial growth?
What is a disinfectant?
a chemical substance that do not sterilize, but instead lower the microbial counts on surfaces
What is an antiseptic?
chemical substance that does not sterilize, but instead lower the microbial counts on skin
What is the effectiveness of disinfectants related to?
2. time of contact
3. physical conditions (temp, humidity, pH, presence of organic matter)
What groups of disinfectants did we test?
1. phenolics (lysol)
2. alcohols (not 100% - needs water)
4. heavy metals
5. halogens (hydrogen peroxide)
What type of organisms is most affected by disinfectant?
gram-positive, smaller affect on gram negative bacteria
What is the phenol coefficient?
60-95% alcohol works best; 100% alcohols are not effective because water is needed to denature the proteins)
What pathogen will Mueller-Hinton-Tellurite agar test for?
Corynebacteria diptheriae - appear as black pin point growths
What does gamma hemolysis on a blood agar tell us?
no lysis of RBC, no change in appearance of the medium=not strep
What does alpha hemolysis on a blood agar tell us?
incomplete lysis of RBC, greenish halo around the bacterial growth
What does beta hemolysis on a blood agar tell us?
lysis of RBC with complete destruction results in a clear zone surrounding the colonies
Mannitol Salt agar: what does it test for and what is a positive result?
1. finds if a bacteria can ferment mannitol
2. agar changes from red to yellow zone around the bacteria for a positive result
MacConkey agar: what does it test for and what is a positive result?
1. isolates gram-neg bacteria; test for enteric bacteria that ferments lactose
2. when bacteria pick up red color that makes them look pink and does not change the agar color = positive
Eosin-methylene blue agar: what does it test for and what is a positive result?
permits differentiation between enteric lactose fermenters and nonfermenters
1. E. coli test positive and are blue-black with a metallic green sheen
2. other bacteria that test positive produce thick, mucoid, pink colonies
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