Exercises 6-7

General purpose media

supports growth of variety of organisms and are good for maintaining cultures

What are examples of general purpose media?

• nutrient agar (NA)

• tryptic soy agar (TSA)

• brain heart infusion (BHI)

Selective media

uses INHIBITORS to prevent growth of certain organisms, allowing others to grow

What are examples of selective media?

• phenyl ethyl agar (PEA)

• eosin methylene blue (EMB)

• macconkey agar

Differential media

allows growth of many microbes but uses INDICATORS to show visible differences between them

What indicators might differential media use?

• dyes

• reagents

• blood cells

• culture conditions

What are examples of differential media?

• eosin methylene blue (EMB)

• blood agar

• macconkey agar

Combination media

media that are both selective and differential

Complex media

are undefined media where the exact chemical composition is not known

How are nutrients supplied in complex media?

as extracts or digests from natural sources

What are examples of complex media?

• brain heart infusion (BHI)

• tryptic soy agar (TSA)

Defined media

are media with a known chemical composition for each component

Is phenylethyl alcohol agar (PEA) selective, differential, or both?

selective only

What is the inhibitor in PEA agar?

phenylethyl alcohol

What type of bacteria does PEA inhibit?

gram-neg bacteria

What type of bacteria does PEA select for?

gram-pos bacteria

What is PEA often used for in patient samples?

to isolate gram-positive bacteria such as staphylococci and eliminate contaminants like E.coli

Is blood agar (BA) selective, differential, or both?

differential only

What is the indicator in blood agar?

red blood cells

What is the base and additive used to make blood agar?

tryptic soy agar (TSA) is the base with 5% sheep’s red blood cells added

What does blood agar differentiate between?

hemolysis patterns - the lysis of red blood cells by microorganisms

What are the types of hemolysis observed on blood agar?

• beta

• alpha

• gamma

Is MacConkey agar selective, differential, or both?

both selective and differential

• its a combination medium

What are the indicators in MacConkey agar?

neutral red

• differentiates lactose fermenters from non-fermenters

What are the inhibitors in MacConkey agar?

crystal violet and bile salts

What do the inhibitors in MacConkey agar do?

prevents growth of gram-pos bacteria

What does MacConkey agar differentiate between?

lactose fermenters (which produce pink colonies) and non-lactose fermenters (colorless colonies)

What type of organisms is MacConkey agar used to identify?

gram-negative enterics

• Enterobacteriaceae

Is Eosin Methylene Blue (EMB) agar selective, differential, or both?

both selective and differential

• a combination medium

What are the inhibitors in EMB agar?

eosin and methylene blue

What do the inhibitors in EMB agar do?

prevents growth of gram-positive bacteria

What are the indicators in EMB agar?

eosin and methylene blue

• also act as pH indicators

What does EMB agar differentiate between?

lactose fermenters (which produced colored colonies) and non-lactose fermenters (colorless colonies)

What is EMB agar commonly used to screen for?

coliforms

• specifically fecal coliforms

What type of clinical testing can EMB agar be used for?

urinary tract infection (UTI) testing

What allows bacteria to be motile?

the presence of flagella

How can bacterial motility be tested in labs?

using motility media

What makes motility detectable in motility media?

the use of soft agar with a decreased agar concentration that allows bacteria to move through it

What is sometimes added to motility media to help visualize movement?

a dye indicator, such as tetrazolium chloride

How is motility media inoculated?

with a single line stab straight down the center of the medium

What indicates a motile organism in motility media?

growth spreading away from the inoculation stab line 

• diffused, cloudy appearance

What indicates a non-motile organism in motility media?

growth that remains confined to the stab line

What is the purpose of the viable plate count method?

to dilute a broth culture so that individual cells can be isolated and counted after plating

What technique is commonly used in a viable plate count?

the spread plate technique

What happens when colonies grow on the viable plate count?

can be counted to determine the original concentration of viable cells in the sample

What are viable cells?

cells that can replicate and form colonies

What type of cells does the viable plate count not detect?

dead cells

What is the formula for the dilution factor?

A/(A+B)

In the dilution factor formula, what does A represent?

the volume being transferred

In the dilution factor formula, what does B represent?

the volume being added to

What is total tube dilution?

the total dilution of the original sample in each tube

How do you calculate the total tube dilution?

multiply the combined dilution of the previous tube by the dilution factor of the current transfer

What is the formula for plate dilution?

plate dilution = (amount plated in mL)/1mL

After incubation, what plates should be counted in a standard plate count?

plates with 30-300 colonies

What must be calculated between the counted plate and the original culture?

the dilution factors (DF)

How is the original concentration of bacteria determined?

multiply the colony count by the reciprocal (inverse) of the dilution factor

How are the results of a standard plate count reported?

CFU/mL

• colony-forming units per mililiter