LABORATORY EXPLORATION # 1

Streaking for Isolation and Interpreting Primary Culture Results

Streaking for Isolation and Interpreting Primary Culture Results

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 Plate Media:
Inoculating loop 4 BAP
Bacti-Incinerator 4 MaC
Broth cultures of: 2 Choc
1. Staphylococcus aureus 2 CNA
2. Escherichia coli Sterile swabs
3. Streptococcus sp.
4. Proteus sp.

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
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
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 LABORATORY EXPLORATION # 1
Streaking for Isolation and Interpreting Primary Culture Results

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.
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)

o Supports the growth of a wide range of organisms
 MaC = MacConkey Plate (selective and differential)
o Selective for gram negative bacilli and differential for lactose fermentation
 Choc(CA) = Chocolate Plate (nutrient)
o 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 = Thioglycollate Broth (nutrient)
 ana = Anaerobic
 CNA = Columbia Colistin-Nalidixic acid
o Selective for gram positive organisms
o Campy = Campylobacter (selective)
o TM= Thayer Martin
 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.
Procedure:

1. Turn on the Bacti-Incinerator and allow to heat until the heating element turns red hot.
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 LABORATORY EXPLORATION # 1
Streaking for Isolation and Interpreting Primary Culture Results

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.
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 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.
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.
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 LABORATORY EXPLORATION # 1
Streaking for Isolation and Interpreting Primary Culture Results

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.

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.

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
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 LABORATORY EXPLORATION # 1
Streaking for Isolation and Interpreting Primary Culture Results

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 10 5 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.

Step 1: Step 2:

Results Form

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 Instructor
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 LABORATORY EXPLORATION # 1
Streaking for Isolation and Interpreting Primary Culture Results

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
11. Proteus on MAC

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 LABORATORY EXPLORATION # 1
Streaking for Isolation and Interpreting Primary Culture Results

Name:___________________________
Date: ___________________________

Study Questions

1. Define the various types of media AND give an example.

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

3. The agar in a nutrient agar serves as a _________________________.

4. Why is a loop flamed before it is placed into a specimen?

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

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