FACULTY OF ENGINEERING TECHNOLOGY

DEPARTMENT OF CHEMICAL ENGINEERING TECHNOLOGY

CELL AND TISSUE ENG. TECH. LABORATORY

LABORATORY INSTRUCTION SHEETS

COURSE CODE BNN 30104

EXPERIMENT CODE

EXPERIMENT TITLE Streak method & technique of transferring culture

DATE

STUDENT NAME & MATRIK NO.

GROUP

GROUP MEMBERS

LECTURER/ INSTRUCTOR/
TUTOR

DATE OF REPORT SUBMISSION

MARKS: ATTENDANCE/DICIPLINE: /5%

RESULTS: /25%

DATA ANALYSIS: /25%

DISCUSSION & CONCLUSION: /35%

REFERENCE: /10%

TOTAL: /100%

EXAMINER COMMENTS: RECEIVED DATE AND STAMP

 KOD ETIKA PELAJAR
(KEP)
JABATAN TEKNOLOGI KEJURUTERAAN KIMIA

FAKULTI TEKNOLOGI KEJURUTERAAN

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Ahli 1 Nama:

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Ahli 2 Nama:

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Ahli 4 Nama:

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___________________________

Tandatangan Pelajar

Nama : _______________________________

No. Matrik :____________________________

Tarikh :________________________________
 FACULTY: ENNGINEERING EDITION:
TECHNOLOGY 1

LABORATORY: CELL AND TISSUE

REVISION NO:
ENG TECH LABORATORY

EXPERIMENT: Streak method & EFFECTIVE DATE: 18/2/2014

technique of transferring culture AMENDMENT DATE:

1.0 OBJECTIVE
The objectives of this experiment are to:
1. perform and explain the streak method
2. perform and explain the technique of transferring culture aseptically

2.0 LEARNING OUTCOMES

i) The student will be able to learn and practice sterile techniques used to culture
bacteria
ii) The student will be able to understand the procedure to obtain isolated microbial
colonies from an inoculums.

3.0 INTRODUCTION / THEORY

Bacterial cultivation technique involves the technique of transferring inoculums. Inoculums is

a small amount of microorganisms used in cultivation to produce more microorganisms and
product. Inoculum transfer is performed aseptically using sterile loop. In this experiment,
student will be exposed to several standard techniques of bacteria cultivation.

The streak plate method is a rapid qualitative isolation method. The techniques commonly
used for isolation of discrete colonies initially require that the number of organisms in the
inoculums be reduced. It is essentially a dilution technique that involves spreading a loopful of
culture over the surface of an agar plate. The resulting diminution of the population size
ensures that, following inoculation, individual cells will be sufficiently far apart on the surface
of the agar medium to effect a separation of the different species present. Although many type
of procedures are performed, the four ways or quadrant streak is mostly done.

The human body has billions of bacteria which constitutes the normal flora fighting against the
invading pathogens. It is tedious to isolate a particular type of bacteria from a clinical sample.
Streak plate technique is used to grow bacteria on a growth media surface so that individual
bacterial colonies are isolated and sampled. Isolated colonies indicate a clone of cells, being
derived from a single precursor cell. When the selected culture media is inoculated using a
single isolated colony, the resulting culture grows from that selected single clone. The modern
streak plate method has evolved from the efforts by Robert Koch and other microbiologists to
obtain pure culture of bacteria in order to study them. The dilution or isolation by streaking
procedure was originally developed by Loeffler and Gaffky in Koch's laboratory, which involves
the dilution of bacteria by systematically streaking them over the surface of the agar in a petri
dish to obtain isolated colonies which will subsequently grow into mass of cells, or isolated
colonies. If the agar surface grows microorganisms which are all the genetically same, the
culture is then considered as a pure culture.

The commonly used petri dishes are of hundred millimetre diameter. The agar surface of the
plate should be dry without any moisture such as condensation drops. The source of
inoculums can be clinical specimen, environmental swab, sedimented urine, broth or solid
culture.

In the streaking procedure, a sterile loop or swab is used to obtain an uncontaminated

microbial culture. The process is called "picking colonies" when it is done from an agar plate
 FACULTY: ENNGINEERING EDITION:
TECHNOLOGY 1

LABORATORY: CELL AND TISSUE

REVISION NO:
ENG TECH LABORATORY

EXPERIMENT: Streak method & EFFECTIVE DATE: 18/2/2014

technique of transferring culture AMENDMENT DATE:

with isolated colonies and is transferred to a new agar or gelatin plate using a sterile loop or
needle. The inoculating loop or needle is then streaked over an agar surface. On the initial
region of the streak, many microorganisms are deposited resulting in confluent growth or the
growth of culture over the entire surface of the streaked area. The loop is sterilized by heating
the loop in the blue flame of the Bunsen burner, between streaking different sections, or zones
and thus lesser microorganisms are deposited as the streaking progresses. The streaking
process will dilutes out the sample that was placed in the initial region of the agar surface.
There are two most commonly used streak patterns, a three sector "T streak " and a four
quadrant streak methods.

3.1 STEAK METHOD

3.1.1 APPARATUS & MATERIALS

1 inoculum loop

1 nutrient agar plate containing bacteria

1 nutrient agar plate

1 Bunsen burner

1 Marking pen

3.1.2 PROCEDURE

All the process is done in a laminar air flow cabinet aseptically.

a. Label a Petri dish:

Petri dishes are labelled on the bottom rather than on the lid. Write close to the edge of
the bottom of the plate to preserve area to observe the plate after it has incubated.
Labels usually include the organism name, type of agar, date, and the plater's name or
initials. Using sterile cotton swabs, remove any visible water on the agar in the plate or
around the inner rim of the petri plate. Observe the plate and mentally divide it into
three sectors. The plate will then be turned clockwise (if you are right handed) with the
agar side up. The second sector will then be at the top for streaking and then the plate
is turned again so that the third sector can be streaked.
 FACULTY: ENNGINEERING EDITION:
TECHNOLOGY 1

LABORATORY: CELL AND TISSUE

REVISION NO:
ENG TECH LABORATORY

EXPERIMENT: Streak method & EFFECTIVE DATE: 18/2/2014

technique of transferring culture AMENDMENT DATE:

b. Sterilize the Transfer Loop before Obtaining a Specimen:

To streak a specimen from a culture tube, metal transfer loops are first sterilized by
flaming the wire loop held in the light blue area of a Bunsen burner just above the tip
of inner flame of the flame until it is red-hot. If a hot incinerator is available, the loop
may be sterilized by holding it inside the incinerator for 5 to 7 seconds. Once sterile,
the loop is allowed to cool by holding it still. Do not wave it around to cool it or blow on
it. When manipulating bacteria, transfer loops are usually held like a pencil. If plastic
disposable loops are being utilized, they are removed from the packaging to avoid
contamination and after being used, are discarded into an appropriate container. A
new loop is recommended for each sector of an isolation streak plate.

c. Open the culture tube and collect a sample of specimen using the sterile loop:

Isolation can be obtained from any of a variety of specimens. This protocol describes
the use of a mixed broth culture, where the culture contains several different bacterial
species or strains. The specimen streaked on a plate could come in a variety of forms,
such as solid samples, liquid samples, and cotton or foam swabs. Material containing
possibly infectious agents should be handled appropriately in the lab using bio safety
procedure.

Remove the test tube cap. It is recommended that the cap be kept in your right hand
(the hand holding the sterile loop). Curl the little finger of your right hand around the
cap to hold it or hold it between the little finger and third finger from the back. Modern
test tube caps extend over the top of the test tube, keeping the rim of the test tube
sterile while the rim of the cap has not been exposed to the bacteria. The cap can also
be placed on the disinfected table, if the test tube is held at an angle so that air
contamination does not fall down into the tube. Insert the loop into the culture tube
and remove a loopful of broth. Replace the cap of the test tube and put it back into the
test tube rack.

d. Streak the Plate:

The lid of the agar plate has to be opened just sufficiently enough to streak the plate
with the inoculation loop. Minimize the amount of agar and the length of time the agar
is exposed to the environment during the streak process.

Three Sector Streak (t streak):

1. Sterilize the wire loop.

2. Cool the loop by touching it on the edge of the sterile agar plate.
3. Dip the loop into the broth culture containing the mixture of bacteria.
4. Lift the lid of the plate just enough to insert the loop. Drag the loop over the
surface of the top one-third of the plate back and forth in a "zig-zag" formation.
5. The loop has picked up thousands of bacteria which are spread out over the
surface of the agar.
6. Sterilize the loop in the flame.
 FACULTY: ENNGINEERING EDITION:
TECHNOLOGY 1

LABORATORY: CELL AND TISSUE

REVISION NO:
ENG TECH LABORATORY

EXPERIMENT: Streak method & EFFECTIVE DATE: 18/2/2014

technique of transferring culture AMENDMENT DATE:

7. Turn the plate 90 degrees and drag the loop through the area you have just
streaked two to three times and continue to drag the loop in a "zig-zag"
formation in the remaining half of the plate without touching that area again.
8. Sterilize the loop in the flame.
9. Turn the plate 90 degrees. Repeat the procedure. Drag the loop two to three
times through the area you just streaked, and fill in the remaining area of the
plate (zig-zag formation), being very careful not to touch any of the areas you
previously streaked.
10. Incubate the plate for 24 hours. If you streaked correctly, you will see isolated
colonies in the third sector. The heaviest growth will be in the first sector. There
will be less growth and some isolated colonies in the second sector. The third
area should have the least growth with isolated colonies.

Fig 1 : Example : A three sector T streak of Serratia marcescens grown on TSA

Four Quadrant Streak :

1. Loosen the cap of the bottle containing the inoculum.

2. Hold an inoculation loop in your right hand.
3. Flame the loop and allow it to cool.
4. Lift the test tube containing the inoculum with your left hand.
5. Remove the cap/ cotton wool plug of the test tube with the little finger of your
right hand.
6. Flame the neck of the test tube.
7. Insert the loop into the culture broth and withdraw. At all times hold the loop
as still as possible.
8. Flame the neck of the test tube again.
9. Replace the cap/ cotton wool plug of the test tube using the little finger of your
right hand. Place the test tube in a rack. For a liquid culture, dip the loop into
the broth, or for solid media, lightly touch a colony with the loop.
10. Partially lift the lid of the Petri dish containing the solid medium.
11. Place a loopful of the culture on the agar surface on the area 1. Flame the loop
and cool it for 5 seconds by touching an unused part of the agar surface close
to the periphery of the plate, and then drag it rapidly several times across the
surface of area1.
12. Remove the loop and close the Petri dish.
 FACULTY: ENNGINEERING EDITION:
TECHNOLOGY 1

LABORATORY: CELL AND TISSUE

REVISION NO:
ENG TECH LABORATORY

EXPERIMENT: Streak method & EFFECTIVE DATE: 18/2/2014

technique of transferring culture AMENDMENT DATE:

13. Reflame and cool the loop, and turn the petri dish 90C then touch the loop to
a corner of the culture in area1 and drag it several times across the agar in
area 2, hitting the original streak a few times. The loop should never enter area
1 again.
14. Remove the loop and close the Petri dish.
15. Reflame and cool the loop and again turn the dish 90C anticlockwise. streak
area 3 in the same manner as area 2, hitting last area several times.
16. Remove the loop and close the Petri dish.
17. Flame the loop, again turn the dish 90C and then drag the culture from a
corner of a area3 across area 4, contacting area 3 several times and drag out
the culture as illustrated. Using a wider streak. Do not let the loop touch any of
the previously streaked areas. The flaming of the loop at the points indicated is
to effect the dilution of the culture so that fewer organisms are streaked in each
area, resulting in the final desired separation.
18. Remove the loop and close the Petri dish.
19. Tape the plate closed and incubates the plate in an inverted position in an
incubator for 24-48 hours.
20. Flame the loop before putting it aside.

Fig 2: Quadrant Streak Method

3.2 TECHNIQUE OF TRANSFERRING CULTURE FROM AGAR PLATE TO BOTTLE

CONTAINING BROTH
 FACULTY: ENNGINEERING EDITION:
TECHNOLOGY 1

LABORATORY: CELL AND TISSUE

REVISION NO:
ENG TECH LABORATORY

EXPERIMENT: Streak method & EFFECTIVE DATE: 18/2/2014

technique of transferring culture AMENDMENT DATE:

3.2.1 APPARATUS & MATERIALS

1 inoculation loop
1 nutrient agar plate containing cultured bacteria
1 bottle containing of LB or nutrient broth
1 Bunsen burner

3.2.2 PROCEDURES

1. Sterile the inoculation loop by replacing it over the flame and let it cool for a while.
Follow the instruction as in part 3.1.2 (b)
2. Remove the bottle lid using your left index finger and expose the bottle mouth over the
flame of Bunsen burner to sterilize it
3. Transfer the bacteria culture on the inoculation loop into the broth. Homogenize it
carefully and aseptically
4. Expose the bottle mouth over the flame again and close the lid
5. Mark this bottle and incubate it at 30C for 24-48 hours.

4.0 RESULTS & CALCULATIONS / ANALYSIS

1. Show the picture for each of the steak method.

2. Explain your findings.

3. State the systematic bias error that could occur during this experiment.

5.0 DISCUSSION AND CONCLUSION

Please provide discussion and conclusion for your findings.

6.0 QUESTIONS

Please answer questions below:

 FACULTY: ENNGINEERING EDITION:
TECHNOLOGY 1

LABORATORY: CELL AND TISSUE

REVISION NO:
ENG TECH LABORATORY

EXPERIMENT: Streak method & EFFECTIVE DATE: 18/2/2014

technique of transferring culture AMENDMENT DATE:

1. During streak plate technique, the loop is heated in between the sector of quadrants.
This ensures:

a) To get dense colonies

b) To get individual isolated colonies
c) To differentiate Gram positive and Gram negative bacteria
d) To kill the infectious microorganism

2. Streak plate is a

a)Qualitative isolation method

b) Quantitative isolation method
c)Non dilution isolation method
d) None of the above

3. A pure culture is best defined as

a) group of microorganisms that are genetically similar

b) group of microorganisms which are genetically different
c) group of microorganisms which are genetically similar, but
morphologically different
d) None of the above

4. To determine the size, colony morphology and grouping of bacterial cells, we use

a) Streak plate phenol red

b) simple stain nutrient broth culture

5. The order of growth seen in the first, second, third and the fourth quadrant of a four
quadrant streak plate

a) Weak growth, heavy confluent growth, dense growth, isolated

colonies
b) Heavy confluent growth, dense growth, weak growth and isolated
colonies
c) Dense growth, heavy confluent growth, weak growth , isolated
colonies
d) All of the above

6. You are given with a "pure" culture of E. coli.

a. What can you do to verify its purity?
b. If the culture was not pure what would you notice?

7. When you observed your streak plate there was lot of undesired colonies growing in it?
8. What might be the common mode of contamination?
 FACULTY: ENNGINEERING EDITION:
TECHNOLOGY 1

LABORATORY: CELL AND TISSUE

REVISION NO:
ENG TECH LABORATORY

EXPERIMENT: Streak method & EFFECTIVE DATE: 18/2/2014

technique of transferring culture AMENDMENT DATE:

9. What is meant by a Bacterial Colony?

10. What is meant by Bacterial Growing Media?
11. After streak plate is done the plates are incubated upside down. What happens if it is
kept upright for incubation?

7.0 REFERENCE

Cid, A. G., and V. B. Rajal. 2011. New teaching strategies to improve student performance in
fundamentals of biotechnology. J. Microbiol. Biol. Educ. 12:4647.

Marintcheva, B. 2012. Motivating students to learn biology vocabulary with Wikipedia. J.

Microbiol. Biol. Educ. 13:6566.

Rosalie J. Cot, 1998. Aseptic Technique for Cell Culture, Current Protocols in Cell Biology,
Current Protocols in Cell Biology. Part 1.3.1-1.3.10 by John Wiley & Sons, Inc. Becton
Dickinson Microbiology Systems Sparks, Maryland,

Chatigny, M.A. 1986. Primary barriers. In Laboratory Safety: Principles and Practices (B.M.
Miller, D.H.M. Grschel, J.H. Richardson, D.Vesley, J.R. Songer, R.D. Housewright, and W.E.
Barkley, eds.) pp. 144-163. American Society for Microbiology, Washington, D.C. Freshney, R.I.
1994. Culture of Animal Cells: A Manual of Basic Technique, 3rd ed., pp. 51-69. Wiley-Liss,
New York.

Virtual Amrita Laboratories Education. Accessed date: 1 February 2015. Link:

http://amrita.vlab.co.in/?sub=3&brch=73&sim=213&cnt=1

Prepared by / Disahkan oleh: Approved by / Disahkan oleh :

Signature/Tandatangan:
Signature / Tandatangan :
Name/Nama: Dr Norshuhaila Mohamed Sunar
Name / Nama : Prof. Madya Dr. Ishak Baba
Date/Tarikh : Date / Tarikh :