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
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
MLAB 2534 – Laboratory 1 – Page 1
LABORATORY # 1
Streaking for Isolation and Interpreting Primary Culture Results
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 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 2
LABORATORY # 1
Streaking for Isolation and Interpreting Primary Culture Results
Primary Plating Media for Common Clinical Samples
Specimen
Routine Media
Throat
BAP, Choc
Sputum
BAP, MaC, Choc
Urine
BAP, MaC
Stool/rectal
BAP, MaC, HE, Sel F, Campy, CNA
Cerebrospinal fluid
BAP, MaC, Choc, Thio
& other body fluids
Cervical, vaginal,
BAP, TM, MaC, Thio
urethral
Abscess, wounds
BAP, MaC, Choc, ana , Thio
Eye, ear
BAP, MaC, Choc, Thio
Skin, pustules
BAP, MaC, Choc, Thio










Temperature/Atmosphere
35 º C, CO2
35 º C, CO2
35 º C
35 º C
35 º C, CO2
35 º C, CO2
35 º C, CO2
35 º C, CO2
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 = Thioglycollate Broth (nutrient)
ana = Anaerobic
CNA = Columbia Colistin-Nalidixic acid
 Selective for gram positive organisms
Campy = Campylobacter (selective)
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.
MLAB 2534 – Laboratory 1 – Page 3
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 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.
MLAB 2534 – Laboratory 1 – Page 4
LABORATORY # 1
Streaking for Isolation and Interpreting Primary Culture Results
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.
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
MLAB 2534 – Laboratory 1 – Page 5
LABORATORY # 1
Streaking for Isolation and Interpreting Primary Culture Results
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
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 6
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 7
LABORATORY # 1
Streaking for Isolation and Interpreting Primary Culture Results
Step 1:
Step 2:
MLAB 2534 – Laboratory 1 – Page 8
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
Instructor
Approval
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 9
LABORATORY # 1
Streaking for Isolation and Interpreting Primary Culture Results
Plate (2 pts. Ea.)
Amt. of Growth
Colony Morphology
Instructor
Approval
11. Proteus on MAC
MLAB 2434: Laboratory #1
Results Form
Points= 2
Name ______________________________________________ Date ___________________________________
MLAB 2534 – Laboratory 1 – Page 10
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.)
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 11