lab 14 - isolation/identification of an unknown bacteria

how do we identify an etiologic agent (the cause of an infectious disease)?

1. isolate the organism in pure culture to eliminate other potential pathogens (do this by streaking for isolation)

2. take growth from a single CFU and aseptically transfer it to grow in pure culture

3. incubate, and now it is the stock culture which is used for all future testing

what is selective medium/agar?

media that contains inhibitory agents that inhibit the growth of specific microorganisms, thereby allowing for the growth of only other specific microorganisms

what is differential agar media?

media that provides preliminary results that logically direct the next biochemical tests and the ultimate definitive identification. most selective agars are also differential

what is macconkey agar?

a medium that is commonly used to select for the growth of gram-negative bacteria and to differentiate those organisms according to their ability to ferment lactose

organisms that grow on macconkey agar are

bile-tolerant and crystal violet tolerant, so they can grow in the presence of inhibitory agents

what is columbia cna agar?

medium that is used to select for the growth of gram-positive bacteria

organisms that grow on cna agar are

resistant to the antibiotics colistin and naladixic acid. they can also lyse erythrocytes through hemolysis, and that is how differentiation is often observed

for macconkey’s agar…

1. what are the inhibitory agents?

2. it tests for the selective growth of…

3. what are the differential agents?

4. differential activity?

5. what is the indicator?

6. differential reaction?

1. bile salts, crystal violet

2. gram-negative bacteria

3. lactose

4. lactose fermentation to acid

5. neutral red

6. pink/red colonies (acid) = lactose fermented; colorless/tan colonies (neutral/alkaline) = non-lactose fermenters

for columbia blood agar (cna)…

1. what are the inhibitory agents?

2. it tests for the selective growth of…

3. what are the differential agents?

4. differential activity?

5. what is the indicator?

6. differential reaction?

1. colistin, naladixic acid

2. some gram-positive bacteria

3. RBCs

4. hemolysis of RBCs

5. none

6. gamma, alpha, or beta hemolysis

for mannitol salt agar (msa)…

1. what are the inhibitory agents?

2. it tests for the selective growth of…

3. what are the differential agents?

4. differential activity?

5. what is the indicator?

6. differential reaction?

1. 7.5% NACl

2. salt-tolerant bacteria (mainly staphylococcus)

3. mannitol

4. mannitol fermentation to acid

5. phenol red

6. yellow colonies & agar (acid) = mannitol fermented

for bile esculin agar…

1. what are the inhibitory agents?

2. it tests for the selective growth of…

3. what are the differential agents?

4. differential activity?

5. what is the indicator?

6. differential reaction?

1. bile salts

2. enterococcus & gram-negative enteric bacteria

3. esculin

4. esculin hydrolysis by esculinase

5. ferric citrate

6. blackening of medium = esculin hydrolysis

for desoxycholate agar (DOC)…

1. what are the inhibitory agents?

2. it tests for the selective growth of…

3. what are the differential agents?

4. differential activity?

5. what is the indicator?

6. differential reaction?

1. desoxycholate & citrate salts

2. gram-negative enteric bacilli

3. lactose & peptone source of amino acids

4. lactose fermentation to acid; amino acid metabolism to alkaline

5. neutral red

6. pink/red colonies (acid) = lactose fermented = fecal coliforms; colorless/tan colonies (alkaline) = NLF = non-coliforms

what is the carbohydrate fermentation series tests?

its the 1st step in the biochemical identification of gram-negative bacteria. most organisms use carbohydrates as the primary nutrient catabolized to provide glycolytic substrates and energy.

_ is often the preferred carbohydrate

glucose. however many organisms alternatively use lactose and/or sucrose. some metabolize these organisms through aerobic respiration, but many use fermentative metabolism. the end products vary, but fall into 2 categories: acidic end products and non-acidic end products. gas (such as O2) can be produced as a by-product

in the carbohydrate tests, how are organisms identified?

by the types of carbohydrates they ferment and whether they produce an acidic end product with/without the production of gas (collected in an inverted durham tube)

in the carbohydrate tests, what is the indicator?

phenol red. if the organism can ferment the carbohydrate to acid, the indicator will change from red to yellow

on macconkey’s agar, cna agar, and the carbohydrate fermentation test, is escherichia coli…

1. gram positive or negative?

2. growth/no growth & appearance on macconkey?

3. interpretation of results on macconkey?

4. growth/no growth & appearance on cna?

5. interpretation of results on cna?

6. results in glucose, lactose, and sucrose?

7. observations from carbohydrate fermentation tests?

1. negative

2. growth, pink colonies

3. lactose fermenter

4. no growth

5. salt-sensitive

6. for all, produced acid and fermented sugar; CO2 was captured in durham tube

7. same as 6

on macconkey’s agar, cna agar, and the carbohydrate fermentation test, is enterobacter aerogenes…

1. gram positive or negative?

2. growth/no growth & appearance on macconkey?

3. interpretation of results on macconkey?

4. growth/no growth & appearance on cna?

5. interpretation of results on cna?

6. results in glucose, lactose, and sucrose?

7. observations from carbohydrate fermentation tests?

1. negative

2. growth, pink mucoid colonies

3. lactose fermenter

4. no growth

5. salt-sensitive

6. for all, produced acid and fermented sugar; CO2 was captured in durham tube

7. same as 6

on macconkey’s agar, cna agar, and the carbohydrate fermentation test, is salmonella enterica…

1. gram positive or negative?

2. growth/no growth & appearance on macconkey?

3. interpretation of results on macconkey?

4. growth/no growth & appearance on cna?

5. interpretation of results on cna?

6. results in glucose, lactose, and sucrose?

7. observations from carbohydrate fermentation tests?

1. negative

2. growth, colorless colonies

3. non-lactose fermenter

4. no growth

5. salt-sensitive

6. for glucose and sucrose, produced acid and fermented sugar; CO2 was captured in durham tube. for lactose, sugar wasn’t fermented so phenol red remained red/orange

7. doesn’t ferment lactose

on macconkey’s agar, cna agar, and the carbohydrate fermentation test, is serratia marcescens…

1. gram positive or negative?

2. growth/no growth & appearance on macconkey?

3. interpretation of results on macconkey?

4. growth/no growth & appearance on cna?

5. interpretation of results on cna?

6. results in glucose, lactose, and sucrose?

7. observations from carbohydrate fermentation tests?

1. negative

2. growth, colorless/slightly pink colonies

3. non-lactose fermenter

4. no growth

5. salt-sensitive

6. for glucose, produced acid and fermented sugar; CO2 was captured in durham tube. for lactose and sucrose, produced acid and fermented sugar without gas.

7. ferments all sugars

on macconkey’s agar, cna agar, and the carbohydrate fermentation test, is pseudomonas fluorescens…

1. gram positive or negative?

2. growth/no growth & appearance on macconkey?

3. interpretation of results on macconkey?

4. growth/no growth & appearance on cna?

5. interpretation of results on cna?

6. results in glucose, lactose, and sucrose?

7. observations from carbohydrate fermentation tests?

1. negative

2. growth, colorless colonies

3. non-lactose fermenter

4. no growth

5. salt-sensitive

6. for all, didn’t ferment sugar so phenol red remained red/orange

7. is alkaline

on macconkey’s agar, cna agar, and the carbohydrate fermentation test, is neisseria sicca…

1. gram positive or negative?

2. growth/no growth & appearance on macconkey?

3. interpretation of results on macconkey?

4. growth/no growth & appearance on cna?

5. interpretation of results on cna?

6. results in glucose, lactose, and sucrose?

7. observations from carbohydrate fermentation tests?

1. negative

2. no growth

3. fastidious organism

4. no growth

5. salt-sensitive

6. for glucose, produced acid and fermented sugar without gas. for lactose and sucrose, sugar wasn’t fermented so phenol red remained red/orange

7. only ferments glucose

on macconkey’s agar, cna agar, and the carbohydrate fermentation test, is staphylococcus epidermidis…

1. gram positive or negative?

2. growth/no growth & appearance on macconkey?

3. interpretation of results on macconkey?

4. growth/no growth & appearance on cna?

5. interpretation of results on cna?

6. results in glucose, lactose, and sucrose?

7. observations from carbohydrate fermentation tests?

1. positive

2. no growth

3. can’t tolerate bile salts & crystal violet

4. growth, small white colonies

5. not sensitive to salt

6. for glucose and sucrose, produced acid and fermented sugar without gas. for lactose, didn’t ferment sugar so phenol red remained red/orange

7. can only ferment glucose and sucrose

on macconkey’s agar, cna agar, and the carbohydrate fermentation test, is enterococcus durans…

1. gram positive or negative?

2. growth/no growth & appearance on macconkey?

3. interpretation of results on macconkey?

4. growth/no growth & appearance on cna?

5. interpretation of results on cna?

6. results in glucose, lactose, and sucrose?

7. observations from carbohydrate fermentation tests?

1. positive

2. no growth

3. can’t tolerate bile salts & crystal violet

4. growth, small grayish colonies

5. not salt sensitive

6. for all, produced acid and fermented sugar without gas

7. same as 6

on macconkey’s agar, cna agar, and the carbohydrate fermentation test, is staphylococcus aureus…

1. gram positive or negative?

2. growth/no growth & appearance on macconkey?

3. interpretation of results on macconkey?

4. growth/no growth & appearance on cna?

5. interpretation of results on cna?

6. results in glucose, lactose, and sucrose?

7. observations from carbohydrate fermentation tests?

1. positive

2. no growth

3. can’t tolerate bile salts & crystal violet

4. growth, yellow/golden colonies with beta hemolysis

5. not salt sensitive

6. for all, produced acid and fermented sugar without gas

7. same as 6

on macconkey’s agar, cna agar, and the carbohydrate fermentation test, is pseudomonas aeruginosa…

1. gram positive or negative?

2. growth/no growth & appearance on macconkey?

3. interpretation of results on macconkey?

4. growth/no growth & appearance on cna?

5. interpretation of results on cna?

6. results in glucose, lactose, and sucrose?

7. observations from carbohydrate fermentation tests?

1. negative

2. growth, colorless colonies

3. non-lactose fermenter

4. no growth

5. salt sensitive

6. for all, sugar wasn’t fermented so phenol red remained red/orange

7. non-fermenter

difference between presumptive/preliminary and definitive/confirmatory identification?

presumptive - relies on observable characteristics and basic tests to narrow down possibilities. for example, a gram stain indicates whether a bacteria is gram positive or negative

definitive - requires more extensive, specific tests to confirm the species/genus of an organism

compare and contrast presumptive/preliminary and definitive/confirmatory results

purpose

- suggest a possible microorganism identity

- confirm the exact identity of a microorganism

accuracy

- not conclusive

- highly accurate/reliable

speed

- quick

- slow and lengthy

example

- a lactose-fermenting colony on macconkey agar could indicate e. coli

- biochemical testing to confirm it is e. coli

what is selective and differential media and what useful function do they serve?

media that inhibits the growth of some microorganisms while allowing for the growth of others. it is helpful because it selects for a specific group of bacteria

media that allows multiple organisms to grow but contains indicators that show differences between them. it is helpful because it can differentiate between species based on observable changes

1. what does the term enteric mean?

2. what does the prefix entero- mean?

3. what does the suffix -itis mean?

4. what is the meaning of enteritis and what are its signs/symptoms?

1. refers to the intestines, mostly the small intestine

2. intestine

3. inflammation of a body part

4. the inflammation of the small intestine, accompanied by symptoms/signs like abdominal pain, diarrhea, vomiting/nausea, etc

which organisms are lactose fermenters? which are non-lactose fermenters?

lactose fermenters include:

• escherichia coli

• enterobacter aerogenes

non-lactose fermenters include:

• salmonella enterica

• serratia marcescens

• pseudomonas fluorescens

• pseudomonas aeruginosa

which organisms don’t grow on macconkey or cna agar and why?

gram-positive organisms don’t grow on macconkey, and gram-negative organisms don’t grow on cna. this is because macconkey inhibits gram-positive growth and cna inhibits gram-negative growth

a carbohydrate fermentation broth tube that is yellow indicates the presence of

acid production

what type of substance is in both a carbohydrate fermentation broth tube and differential agar media that indicates a certain condition in the media?

phenol red

what is the name of the specific substance added to carbohydrate fermentation broth tubes and which turns yellow under acidic conditions?

phenol red

what is the function of the small, inverted durham tube in the larger carbohydrate fermentation broth tube?

its used to collect gas (if produced) during fermentation