Clinical Microbiology Laboratory

Laboratory Week 3:   Media Usage and Colonial Morphology:

Activities:

Streaking  Plates Demo  

Demo:  Streaking Pattern Used for All Cultures Other Than Urines

Growth only in the first quadrant: Few
Growth in the first and second quadrant:  Moderate
Growth through the third quadrant: Moderately Heavy
Growth through the fourth or fifth quadrant:  Heavy

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Urine Culture Streaking Pattern

Growth only in the initial quadrant: Less than 104 CFU/ml
Growth in the first and second quadrants: 104 - 105 CFU/ml
Growth in the last quadrant: Greater than 105 CFU/ml

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Streaking Plates Activity

Materials:

  • Sterile disposable inoculating needles

  • Swabs for known organisms

  • Media:  BAP (TSA w/5% Sheep Blood), Chocolate agar,  Columbia CNA agar w/ 5% Sheep Blood, SXT Blood agar, Bile Esculin agar, MacConkey agar, EMB agar, Tergitol 7 agar w/ TTC, (Modified) Thayer Martin agar

  1. Collect a set of media plates for each of the known culture swabs given you.

  2. Label each set of plates with the name of the organism, your initials and the date.

  3. Swab the initial area with the known organism on each plate.

  4. Streak for isolation on each plate with your inoculating loop.  You may use the same inoculating loop for the same organism set of plates.

  5. Incubate the chocolate and modified Thayer Martin agar plates in the the candle jar and the rest in the regular incubator at 35.5 + 1.5oC

 

 

 

Media Charts
Complete the following chart using the material from Remel found on pages  54-69 of your Lab Manual
Media Type Grows Inhibits Differentiates
BAP (TSA w/5% Sheep Blood)
 Supportive Most organisms except Neisseria gonorrhoeae & Haemophilus influenzae None Types of Hemolysis
  • Alpha (a)
  • Beta (b)
  • Gamma (g)
Chocolate agar
 Supportive Most organisms including Neisseria gonorrhoeae & Haemophilus influenzae None None
Columbia CNA agar w/ 5% Sheep Blood
 Selective & Differential Gram-positive organisms Gram-negative organisms Types of Hemolysis
  • Alpha (a)
  • Beta (b)
  • Gamma (g)
SXT Blood agar
 Selective & Differential Group A and B b hemolytic streptococci Normal throat flora Types of Hemolysis
  • Alpha (a)
  • Beta (b)
  • Gamma (g)
Bile Esculin agar
 Selective & Differential  

?

 Streptococci not in Group D Esculin Hydrolysis = ________ color
MacConkey agar
 Selective & Differential ? ? Lactose fermenter = ________ color

Nonlactose fermenter = _________ color
EMB agar
 Selective & Differential ? ? Lactose fermenter = ________ color

Nonlactose fermenter = _________ color
Tergitol 7 agar w/ TTC
 Selective & Differential ? ? Lactose fermenter = ________ color

Nonlactose fermenter = _________ color
(Modified) Thayer Martin agar
 Selective ? Most Gram positives and Gram negatives, Has nystatin present but yeast will still grow None

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Media for Specific Culture Specimens
 (Page 51 of Lab Manual)

Urine

Blood

Wound

Genital

CSF

Throat

Naso-
pharynx

Stool

Sputum

Sterile
Body
Fluids

BAP

X

X

X

X

X

X

X

X

X

X

CHOC

-

X

-

X

X

-

X

-

X

X

GNR*
Selective

X

-

X

X

(X)

-

-

X

X

-

SXT

-

-

-

-

-

X

-

-

-

-

MTM

-

-

-

X

-

(X)

(X)

-

-

-

CNA

(X)

-

X

X

-

-

-

-

-

-

XLD

-

-

-

-

-

-

-

X

-

-

HE

-

-

-

-

-

-

-

X

-

-

CAMPY

-

-

-

-

-

-

-

X

-

-

THIO

-

-

X

-

-

-

-

-

-

-

SMAC

-

-

-

-

-

-

-

X

-

-
  • * This could be MacConkey agar, EMB agar, or Tergitol 7 agar with TTC
  • (X) GNR if Gram Negative Rods are seen in the Gram Stain or if the patient is a newborn or elderly patient.
  • (X) MTM if it is screening for Neisseria meningitidis or Neisseria gonorrhoeae pharyngitis
  • (X) CNA if the Gram stain shows both Gram negative rods and Gram positive organisms.

Complete a worksheet related to Media Charts and Laboratory Manual pages 50 - 68

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Reading Plates of Gram Positive Organisms

  1. Consult page 53 of your lab manual.

  2. Using the blue sheets provided for these organism, record your results.

  3. Read the type of hemolysis present on all the agars containing blood.

  4. Indicate whether each of the organisms grew normally (compare with your blood or chocolate plate), was inhibited, or did not grow.

  5. You will see little or no growth on MacConkey, Tergitol, or EMB for these organisms BUT check for inhibited growth very carefully.

  6. Besides growth or no growth on Bile esculin agar indicate whether the organism hydrolyzed esculin. (brown to black colony with a brown to black halo)
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Reading Plates of Gram Negative Organisms

  1. Consult page 53 of your lab manual.

  2. Using the pink sheets provided for these organisms, record  results from all plates (yours and your classmates).

  3. Read for beta-hemolysis present on all the agars containing blood.

  4. Indicate whether each of the organisms grew normally (compare with your blood or chocolate plate), was inhibited, or did not grow.

  5. You will see growth on MacConkey, Tergitol, or EMB for a number of these organisms.  You will also record whether the organisms are lactose fermenters or appear to be nonlactose fermenters.  (Be sure you know the type of colonies for each.)

  6. Besides growth or no growth on Bile esculin agar indicate whether the organism hydrolyzed esculin. (brown to black colony with a brown to black halo)
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Clinical Microbiology Syllabus