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What are the 3 chemical reagents that are used in differential staining?

1. Primary stain
2. decolorizing agent
3. counterstain

What is the most important differential stain used in bacteriology?

Gram stain

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-postive cells have?

a thick peptidoglycan layer

What kind of cell wall does Gram-negative cells have?

thinner and surrounded by outer lipid-containing layers

What is the primary stain that is used in a Gram stain?

Crystal Violet

What color crystal violet make the stain?


What is the mordant used in a gram stain?

Gram's Iodine

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 is the decolorizing agent used in Gram stain?

Gram decolorizer, or ethyl alcohol, 95%

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 is the counterstain used in Gram stain?


What is the most important step in the gram stain?


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

Know and label the following on a microscope.


Gram stain




tetrad coccus

What shape are cocci?




What shape are Bacilli?


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

TSI - What does a black butt mean?

organism is capable of producing hydrogen sulfide

What are the 3 types of enterics found in the intestinal tract of humans?

1. pathogens
2. occasional pathogens
3. normal intestinal flora

Name some examples of enteric pathogens.

1. Salmonella
2. Shigella

Name some examples of enteric occastional pathogens.

1. Proteus
2. Klebsiella

Name some examples of enteric normal intestinal flora.

1. Escherichia
2. Enterobacter

What 3 types IMViC tests can be done to identify enteric bacteria.

1. indole
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 medium stand for?

hydrogen Sulfide, Indol, and Motility

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?

1. sterilization
2. disinfectants
3. antiseptics
4. preservatives

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?

1. concentration
2. time of contact
3. physical conditions (temp, humidity, pH, presence of organic matter)

Is there a single disinfectant that is effective in all situations?


What groups of disinfectants did we test?

1. phenolics (lysol)
2. alcohols (not 100% - needs water)
3. iodine
4. heavy metals
5. halogens (hydrogen peroxide)

Which disinfectant was most effective?

hydrogen peroxide

Which disinfectant was the least effective?

ethyl alcohol

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 is the most common cause for strep throat?


What is the most common cause for diaper rash?

Candida albicans "Yeast"

What is the common cause for diphtheria?

Corynebacterium diphtheriae

What is the most common cause for skin diseases?

S. aureus

What pathogen will Mueller-Hinton-Tellurite agar test for?

Corynebacteria diptheriae - appear as black pin point growths

What pathogen will Mannitol Salt Agar test for?


What pathogen will Sabouraud Agar test for?

isolates fungi, which includes molds and yeasts

What pathogen will chocolate agar test for?

H. influenzae

What pathogen will Blood agar plate test for?


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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