LABORATORY # 1

Streaking for Isolation and Interpreting Primary Culture Results

Laboratory #1
Streaking for Isolation and Interpreting Primary Culture Results
24 Points

Objectives:
At the end of this activity, the student will be able to:

1. Inoculate (streak) bacterial cultures on specified agar plates using techniques that will
separate the individual bacterial cells to obtain well-isolated colonies on primary
inoculation media.

2. Recognize key colony characteristics.

3. Interpret primary cultures using colony characteristics and gram stains.

4. State the purpose of the various types of media (i.e. selective, nonselective) and classify a
given medium as to its type.

5. Discuss the various incubation conditions used in the clinical lab.

Materials: Sharpie-type marking pen

Inoculating loop
Bacti-Incinerator
Broth cultures of

MLAB 2534 – Laboratory 1 – Page 1

 LABORATORY # 1

Streaking for Isolation and Interpreting Primary Culture Results

1. Staphylococcus aureus
2. Escherichia coli
3. Streptococcus sp.
4. Proteus sp.
4 BAP
4 MaC
2 Choc
2 CNA
Sterile swabs

References:
Mahon and Manuselis, Textbook of Diagnostic Microbiology, Fourth Edition, Chapter 6

Principles:
Isolation of the infecting agent (bacteria) in culture is the most sensitive and specific means of
laboratory diagnosis of infectious diseases. Most bacteria can be cultivated in vitro (outside the
body) using artificial culture media (plural; singular is “medium”). Isolation of bacterial colonies
is essential to work with pure cultures, in order to determine an organism’s colonial
characteristics, biochemical properties, and other details. The primary media selected for
cultivation of organisms depend on the suspected causative bacteria from a particular clinical
sample. Clinical microbiology laboratories use a wide variety of growth media for isolation of
commonly encountered bacterial agents. Primary inoculation is made with a loop, swab, or
other suitable device.

General guidelines:
Liquid specimens- 1-2 drops
Feces or sputum- dip swab into specimen

MLAB 2534 – Laboratory 1 – Page 2

 LABORATORY # 1

Streaking for Isolation and Interpreting Primary Culture Results

Swabs- direct

There are several types of culture media used for specific purposes. They can be grouped into
broth media or agar media. Agar is a polysaccharide extracted from algae which is a solidifying
agent much like gelatin. Agar provides a support matrix and nutrients on which to culture or
grow microorganisms.

These media are classified as: nutrient, selective, and differential or indicator.

A nutrient medium is used primarily to satisfy the growth requirements of bacteria. This
medium supports the growth of most nonfastidious (hardy) organisms. For other pathogens
that require special nutrients for growth, vitamins, salts, and body fluids may be added to the
nutrient base. Examples include TSA: trypticase soy agar or brain heart infusion agar.

Selective media are used when specific significant organisms are to be isolated. Chemical dyes
or antimicrobials (also known as antibiotics) are added to the medium to inhibit contaminating
organisms but not the suspected agent. Examples include PEA and CNA.

Indicator or differential media are designed to demonstrate certain diagnostic features of

specific pathogens. The medium contains an indicator system, such as a pH indicator, and a
carbohydrate, which shows color change in the colony when the carbohydrate is used.
Examples include triple sugar iron agar and MacConkey agar.

Broth medium is liquid and is used as enrichment medium to allow small number of organisms
to grow. Examples include thioglycolate broth or Todd Hewitt.

MLAB 2534 – Laboratory 1 – Page 3

 LABORATORY # 1

Streaking for Isolation and Interpreting Primary Culture Results

Primary Plating Media for Common Clinical Samples

Specimen Routine Media Temperature/Atmosphere
Throat BAP, Choc 35 º C, CO2
Sputum BAP, MaC, Choc 35 º C, CO2
Urine BAP, MaC 35 º C
Stool/rectal BAP, MaC, HE, Sel F, Campy, CNA 35 º C
Cerebrospinal fluid BAP, MaC, Choc, Thio 35 º C, CO2
& other body fluids
Cervical, vaginal, BAP, TM, MaC, Thio 35 º C, CO2
urethral
Abscess, wounds BAP, MaC, Choc, ana , Thio 35 º C, CO2
Eye, ear BAP, MaC, Choc, Thio 35 º C, CO2
Skin, pustules BAP, MaC, Choc, Thio 35 º C, CO2

BAP = Blood Agar Plate (nutrient)

Supports the growth of a wide range of organisms
MaC = MacConkey Plate (selective and differential)
Selective for gram negative bacilli and differential for lactose fermentation
Choc (CA) = Chocolate Plate (nutrient)
Supports the growth of a wide range of organisms, including fastidious ones
HE = Hektoen-Enteric Plate (selective and differential)
Sel F = Selenite F (fecal) Broth (selective)
Thio = Thioglycolate Broth (nutrient)
ana = Anaerobic
CNA = Columbia Colistin-Nalidixic acid
Selective for gram positive organisms
Campy = Campylobacter (selective)
TM= Thayer Martin

MLAB 2534 – Laboratory 1 – Page 4

 LABORATORY # 1

Streaking for Isolation and Interpreting Primary Culture Results

Selective for Neisseria species

**NOTE: Primary plating media will vary depending on the facility and target population. This
chart is provided as a guide to media selection for this course. Bookmark this page for use in
unknown determinations.

MLAB 2534 – Laboratory 1 – Page 5

 LABORATORY # 1

Streaking for Isolation and Interpreting Primary Culture Results

Procedure:

1. Turn on the Bacti-Incinerator and allow to heat until the heating element turns red hot.

2. Obtain a paper template of a petri dish to practice streaking. Perform AT LEAST two (2)
successful streaks on the paper template PRIOR to continuing to step #3. Obtain instructor
approval. Instructor to initial template to be turned in with lab.

3. Working in pairs, label the bottom of agar plates with your initials, date, and either the
organism or patient name. The bottom is the section with the agar. Label according to the
following:
Staphylococcus aureus - BAP, CNA, Mac & Choc
Escherichia coli – BAP & Mac
Streptococcus sp. – BAP, Mac & Choc
Proteus sp.- BAP, Mac, CNA

For each broth culture, remove a sterile swab from its wrapper, holding it between the
thumb and forefinger of the right hand.

4. Next hold one of the broth tubes in the left hand and remove and hold the cap with the little
finger and palm of the right hand.

5. Heat the neck of the broth tube by holding against the opening of incinerator. Dip the swab
into the broth culture tube, saturating it well. Press and swirl the swab against the inside of the
tube above the broth to remove excess. Withdraw the swab, heat the neck of the broth tube
again, and replace the cap. Replace the broth tube in a rack.

6. Remove the agar plate from its lid with the left hand. While holding the plate, inoculate the
agar heavily near the periphery of the plate spanning to approximately ¼ of the agar. This area

MLAB 2534 – Laboratory 1 – Page 6

 LABORATORY # 1

Streaking for Isolation and Interpreting Primary Culture Results

is referred to as the first quadrant (See diagram step #1.) You may use the same swab for each
broth culture to inoculate the first quadrant on several plates, as long as the swab is used to
inoculate the plates in this order: Choc, BAP, MAC. This order is important since it places the
media from least selective to most selective. If a more selective plate is inoculated first, a
potential exists to contaminate the other plates with the antimicrobials from the first plate.

7. Replace the plate into its lid and discard the swab into the appropriate biohazard container.

8. Flame the wire loop and let cool for 2-3 seconds. Read NOTE below.

9. Holding the agar plate with the left hand, streak the original inoculum at a 90° clockwise
angle. (Diagram step #2.) Hold the bacteriological loop loosely between the thumb and index
finger. Allow the weight of the loop to exert its own pressure. There is no need to exert
additional pressure with the hand.

MLAB 2534 – Laboratory 1 – Page 7

 LABORATORY # 1

Streaking for Isolation and Interpreting Primary Culture Results

10. Replace the plate into its lid and flame the loop. Let cool again and streak again clockwise
to and 90 degrees from the second streak. (Diagram step #3.)

11. Repeat step 9, streaking as in diagram step 4.

12. Flame the loop to remove all organisms.

13. Repeat steps 3-12 with the other broth cultures.

14. Incubate at 35° C, CO2 overnight. NOTE: If cultures cannot be examined after 24 hours
(i.e., weekend), arrangements MUST be made for refrigerating plates.

NOTE: Varying methods exist for removing organisms from the loop between quadrants. 
Generally, the metal loop is sterilized between each quadrant by incinerating and then cooling
the loop.  Some clinical microbiologists flame once after the initial quadrant and then rotate the
loop so that the next quadrants can be streaked with an unused side of the loop.  When using
plastic loops, stab the loop several times into the agar to clear the loop between quadrants.  DO
NOT FLAME PLASTIC LOOPS!

Other Inoculation Techniques:

The four (4) corner or quadrant technique will be used in this course, because we are working
primarily with broth cultures with numerous colony forming units (CFU). However, when
working with primary patient cultures, that is, specimens obtained directly from a patient, such
as on a swab, a three (3) corner inoculation or streaking technique is frequently used, since the
number of organisms or CFU found in patient cultures is most often much fewer than those
grown in broth cultures.

MLAB 2534 – Laboratory 1 – Page 8

 LABORATORY # 1

Streaking for Isolation and Interpreting Primary Culture Results

Evaluation of Isolation Technique

After 24 hours incubation, examine the agar plates. There should be sufficient growth of each
organism on the plates and the isolation should be such that in the last corner or quadrant of
growth there should be well-isolated colonies. To determine the amount of growth of each
colony-type, the culture should also be evaluated according to the following semi-quantitative
measures:

Growth in 1st quadrant only = Rare/scant

Growth in 1st and 2nd quadrants only = Few
Growth in 1st, 2nd, and 3rd quadrants = Moderate
Growth in all quadrants = Abundant

Procedure:

1. Using the Colony Morphology handout from the instructor, evaluate the colony morphology
of each culture and include results on the worksheet below. DO NOT throw away your plates,
they will be used for Laboratory #2.
2. If there is no growth on the plate, indicate that on the worksheet by using “NG”
3. Have the instructor review and initial your streaking technique.

MLAB 2534 – Laboratory 1 – Page 9

 LABORATORY # 1

Streaking for Isolation and Interpreting Primary Culture Results

Colony Counts

Determining a colony count:

When inoculating a specimen, primarily urine, which requires a colony (CFU) count, a calibrated
loop is used (usually 0.001 µL). Following incubation, the number of CFU is multiplied by 1000
to correct for volume inoculated and the CFU are reported as CFU or colonies/mL. This
technique will be demonstrated and used later in the course when studying urine cultures.

Although this technique will not be performed for this lab, bookmark the procedure for use
with the course unknowns.

Procedure:
1. Dip a sterile 0.01 mL or 0.001 mL calibrated loop into a well-mixed clean-catch midstream
urine specimen.
2. With the calibrated loop, streak the plate from top to bottom, as shown below.
3. With the same loop, cross streak the primary inoculation at right angles.
4. Repeat for additional plates, as directed.
5. Place in a 35-37 O C incubator overnight.
6. To obtain the number of CFUs per milliliter of urine, multiply the number of colonies on the
plate by the appropriate dilution factor. If a 0.01-mL loop was used, the dilution factor is
100. If a 0.001 loop was used the dilution factor is 1000. For example, if a 0.001-mL
calibrated loop was used, and 300 colonies grew on the plate, the colony count would be
3000 X 1000= 300,000 or 3 x 105 CFU/mL.
7. The final calculated result is reported in CFU/mL.
8. The calculation of CFU/mL must be repeated for each colony type determined to be a
potential pathogen.

MLAB 2534 – Laboratory 1 – Page 10

 LABORATORY # 1

Streaking for Isolation and Interpreting Primary Culture Results

Step 1:

MLAB 2534 – Laboratory 1 – Page 11

 LABORATORY # 1

Streaking for Isolation and Interpreting Primary Culture Results

Step 2:

MLAB 2534 – Laboratory 1 – Page 12

 LABORATORY # 1

Streaking for Isolation and Interpreting Primary Culture Results

MLAB 2534 – Laboratory 1 – Page 13

 LABORATORY # 1

Streaking for Isolation and Interpreting Primary Culture Results

MLAB 2534 – Laboratory 1 – Page 14

 LABORATORY # 1

Streaking for Isolation and Interpreting Primary Culture Results

MLAB 2434: Laboratory #1

Results Form
Points= 24 Total
(2 points paper streaking + 22 points for table)

Name ______________________________________________ Date

___________________________________

Have the instructor examine each plate and check off your technique, along with your colony morphology.
SAVE ALL PLATES FOR FUTURE LABS.

Plate (2 pts. Ea.) Amt. of Growth Colony Morphology Instruc

Appro

1. S. aureus on BAP

2. S. aureus on MaC

3. S. aureus on Choc
4. S. aureus on CNA

4. E. coli on BAP

5. E. coli on MaC

6. Streptococcus on BAP

7. Streptococcus on MaC

8. Streptococcus on Choc
9. Proteus on BAP
10. Proteus on CNA

MLAB 2534 – Laboratory 1 – Page 15

 LABORATORY # 1

Streaking for Isolation and Interpreting Primary Culture Results

11. Proteus on MAC

MLAB 2434: Laboratory #1

Results Form
Points= 2

Name _____________________________________ Date ______________

MLAB 2534 – Laboratory 1 – Page 16

 LABORATORY # 1

Streaking for Isolation and Interpreting Primary Culture Results

Name: ___________________________
Date: ___________________________
Total Points: 10

Lab 1: Study Questions

1. Define the various types of media AND give an example. (4 pts.)

2. Why is it important to have isolated bacterial colonies on a culture plate? (2pts.)

MLAB 2534 – Laboratory 1 – Page 17

 LABORATORY # 1

Streaking for Isolation and Interpreting Primary Culture Results

3. The agar in a nutrient agar serves as a _________________________. (1 pt.)

4. Why is a loop flamed before it is placed into a specimen? (1 pt.)

5. When working with broth cultures, is a three quadrant or four quadrant technique
utilized? Why? (2 pts.)

MLAB 2534 – Laboratory 1 – Page 18