Micro Bio Exam #2

A broth culture containing two Gram-positive and one Gram-negative bacteria for a student unknown ID project is streaked for isolation onto a TSA plate.

•Which of the organisms should form isolated colonies on TSA?

•Which of the plates below shows this result?

-all should form isolated colonies

-Plate A shows this result

A broth culture containing two Gram-positive and one Gram-negative bacteria for a student unknown ID project is streaked for isolation onto a MacConkey plate.

•Which of the organisms should form isolated colonies on Mac?

•Which of the plates below shows this result?

•Which slide shows the results you would get if you Gram negative stained the bacteria growing on this plate?

-Selective and differential media

-selective for gram-negative organism

-plate A shows isolated colonies

-slide A shows gram stained bacteria (pink/red)

A broth culture containing two Gram-positive and one Gram-negative bacteria for a student unknown ID project is streaked for isolation onto a C-CNA plate.

•Which of the organisms should form isolated colonies on C-CNA?

•Which of the plates below shows this result?

-Selective and differential

-contains two antibiotics colistin and nalidixic acid

-selects for gram positive organism (entero/strep)

-Plate B shows isolated colonies

Describe how you would perform a catalase test

-drop 2 drops of 3% hydrogen peroxide onto glass slide

-with a sterile loop. transfer each bacteria onto slide and mix with 3% hydrogen peroxide

staph: pos

strep/entero: neg

Interpret results

catalase neg- no bubbles (streptococcus/enterococcus)

catalase pos- bubbles (staphylococcus)

which plate is this

Staphylococcus tri-plate

-DNase (blue-green)

-Mannitol Salt agar (pink)

-Mueller-Hinton Agar (yellowish-clear)

Is a DNase plate selective, differential or both?

Both

Is the organism on plate A + or - for DNase production

POSITIVE -

due to zone of clearing

Is the organism on plate B + or - for DNase production

NEGATIVE - due to lack of clearing

Give an example of a Staph that is DNase-positive.

Staphylococcus aureus

Give an example of a Staph that is DNase-negative

What would negative look like on plate

Staphylococcus epidermidis

or

Staphylococcus xylosus

Is the organism growing on the Mueller-Hinton sector of plate A susceptible or resistant to Novobiocin?

Susceptible due to clearing around novobiocin disk

Is the organism growing on the Mueller-Hinton sector of plate B susceptible or resistant to Novobiocin?

Resistant - due to no clearing around disk

Give an example of a Staph that is Novobiocin Resistant

Staphylococcus xylosus

Give an example of a Staph that is Novobiocin Susceptible

Staphylococcus aureus

or Staphylococcus epidermis

what makes MSA agar differential?

sugar mannitol - can ferment mannitol (yellow line)

what makes MSA agar selective?

-phenol red

-7.5& NaCl - selects for halotolerant organisms (staph)

Name the pH indicator in the MSA media

phenol red

Is the organism growing on the MSA sector of plate A halotolerant? Does it ferment mannitol?

-not halotolerant

-does not ferment mannitol

Is the organism growing on the MSA sector of plate B halotolerant? Does it ferment mannitol?

-yes halotolerant

-can ferment mannitol

Give an example of a Staph that ferments mannitol

Staphylococcus aureus

or

Staphylococcus xylosus

Give an example of a Staph that does not ferment mannitol

Staphylococcus epidermis

Determine the results for this Staphylococcus tri-plate

Identify the species of staphylococcus using the chart below

MSA: + (yellow)

DNase: + (clearing)

Novobiocin susceptibility: S (35mm)

- Staphylococcus intermedius

Plate A

Hemolysis: Beta

Bacitracin: R

SXT: S

Plate B

Hemolysis: Game

Bacitracin: R

SXT: R

alpha results on blood agar

semi-see through (partial) green color like

beta results on blood agar

see through (complete) yellow color like

gama results on blood agar

no change - only see swab line

Bile Esculin Agar (BEA)

Substrate:

esculin

Bile Esculin Agar (BEA)

color indicator:

Ferric Ammonium citrate

Bile Esculin Agar (BEA)

What are the selective ingredients in this media?

bile salts

Bile Esculin Agar (BEA)

What are the differential ingredients in this media?

esculin and ferric ammonium citrate

Bile Esculin Agar (BEA)

Is tube A positive or negative for esculin hydrolysis?

negative (no color change)

Bile Esculin Agar (BEA)

Is tube B positive or negative for esculin hydrolysis?

Positive (black)

Give an example of a Strep that is positive for esculin hydrolysis.

Streptococcus agalactiae

and

Enterococcus faecalis

Give an example of a Strep that is negative for esculin hydrolysis.

-Streptococcus pyogenes

-Streptococcus Bovis

-Streptococcus Mutans

-Streptococcus equi

-Streptococcus zooepidemicus

An unknown Gram-positive coccus gives a negative catalase reaction but grows on MSA. Name the organism.

enterococcus faecalis

Determine the results for this Strep media:

MSA: pos

Hemolysis: alpha

BEA: +

SXT: R

Bacitracin: R

Enterococcus faecalis

TSI Slant:

What is the pH indication in this:

pH indicator: phenol red

TSI Slant:

2 components of this agar that detects hydrogen sulfide production:

color indicator: ferrous sulfate

substrate: sodium thiosulfate

TSI Slant:

What are the 3 carbohydrates in this medium and what is the % of each

-0.1% Glucose

-1.0% Sucrose

-1.0% Lactose

TSI Slant: Results

What are the results:

Slant:

Butt:

Gas:

H2S:

Slant: Alkaline

Butt: Acidic

Gas: neg

H2S: neg

Ferments: glucose

TSI Slant: Results

What are the results:

Slant:

Butt:

Gas:

H2S:

Slant: acidic

Butt: Acidic

Gas: pos

H2S: pos

Ferments: glucose and lactose/sucrose

TSI Slant: Results

What are the results:

Slant:

Butt:

Gas:

H2S:

Slant: acidic

Butt: Acidic

Gas: pos

H2S: neg

Ferments: glucose and lactose/sucrose

TSI Slant: Results

What are the results:

Slant:

Butt:

Gas:

H2S:

Slant: alkaline

Butt: alkaline

Gas: neg

H2S: neg

TSI Slant: Results

What are the results:

Slant:

Butt:

Gas:

H2S:

Slant: alkaline

Butt: acidic

Gas: neg

H2S: pos

LIA Slant:

Name amino acid

lysine

LIA Slant

What is the pH indicator

bromocresol purple

LIA Slant: lysine deamination

Substrate:

glucose

LIA Slant: lysine deamination

pH indicator:

bromocresol purple

LIA Slant: lysine deamination

color indicator:

ferric ammonium citrate

LIA Slant: lysine decarboxylation

pH indicator:

bromocresol purple

LIA Slant: lysine decarboxylation

substrate:

lysine glucose

LIA Slant: H2S Production

substrate:

sodium thiosulfate

LIA Slant: H2S Production

color indicator:

ferrous sulfate

LIA Slant

Is deamination of lysine an aerobic or anaerobic process?

Where does it occur?

anaerobic process

occurs in the slant

+: red

-: purple

LIA Slant

Is decarboxylation of lysine an aerobic or anaerobic process?

Where does it occur?

anaerobic

occurs in bu**

+: purple

-: yellow

LIA Slant Results

Citrate Slant:

Positive:

Negative:

Positive: Russian blue (A)

Negative: negative - no color change

citrate test tests for ______

citrate (turns tube blue)

Citrate Slant:

pH indicator:

bromthymol blue

Citrate Slant:

Is this medium selective or differential?

both

Determine Results:

Mac fermentation: neg

Urease: + (pink)

citrate: + (blue)

TSI Slant: alkaline

TSI butt: acidic

Gas: neg

H2S: +

Nitrate Broth: (nitrate reduction)

Substrate

Nitrate

Nitrate:

What color indications a positive or negative

+: red

-: no color change or color change after adding zinc

Reagents used in Nirate:

Nitrate A: sulfamic acid

Nitrate B: alpha naphthylamine

Reagents used in TSI:

NONE

Reagents used in Urea:

NONE

Reagents used in SIM (indole production)

Kovac's reagent (HCI, Butanol, para-dimethylaminobenzaldehyde

Reagents used in methyl red broth

methyl red

Reagents used in VP Broth

Barrits A &B

A: aplha naphthol

B: 40% KOH

Reagents used in Citrate:

NONE

Substrate in citrate:

sodium citrate

pH indicator in citrate:

bromthymol blue

Reagents used in LIA:

NONE

SIM Agar Deep h2s production:

substrate

sodium thiosulfate:

SIM Agar Deep h2s production:

Color indicator

ferrous sulfate

SIM interpret results

indole: + red

black prec: + H2s

Growth of line: spreading = positive for motility

1:

indole: +

H2s: -

motility: +

2:

indole: -

H2s:-

motility: +

3:

indole: -

H2s: +

motility: -

4:

indole: -

H2s: -

motility: +

5:

indole: +

H2s: +

motility: +

SIM agar deep

Name amino acid substrate in this medium for indole production

tryptophan

SIM Agar Deep

3 characteristic test for:

H2S, indole, motility

SIM Agar Deep

Motile if ____

growth out from stab line

Urea Broth

Substrate in this media

urea

Urea Broth

pH indicator in this media

phenol red

urea broth result

1: neg

2: pos (pink)

urea broth test for _____

urease

Substrate in MR & VP

glucose

what does MR stand for:

methyl red

what does VP stand for:

Voges-Proskauer

MRVP results

RED: +

yellow/no change: -