Transfer and Colony Selection Techniques & Techniques for Isolating Pure Cultures

Purpose:

To verify presence of bacteria in environment
To be able to perform basic bacteriological transfers
To learn aseptic technique
To become familiar with characteristics of bacterial broth and agar slant cultures
To understand what the pure culture is
To practice spread plating, pour plating and streak plating techniques
To be able to calculate cfu/ml and learn meaning of it
To be able to distinguish basic features of bacterial colonies, broth cultures and agar slant growths
To be able to recognize the advantages and —streak plate— limitations of culture characteristics in the identification of bacterial species

Procedure: Simplified and Combined

Exercise 03

A) Microorganisms in the environment: Take sterile Nutrient agar plates

Test 1: Expose the surface of agar plate to air for

0 min à Group #1
5 min à Group #2
10 min à Group #3
15 min à Group #4
20 min à Group #5

Test 2: Expose the surface of agar plate to followings;

a) Touch the surface of the agar with your fingerà Group #6
b) Combine your hair over agarà Group #7
c) Cough onto agar 1 or 2 timesà Group #8

TRANSFERS*

B) Transfer Broth Escherichia coli culture to sterile NB
C) Use agar slant as a source of inoculum, transfer Bacillus thuringrensis to sterile NB with loop
D) Inoculate a sterile agar slant by using Serratia marcescens broth culture
E) Transfer to an agar deep from Escherichia coli broth culture to sterile deep agar with needle
F) For colony selection use Bacillus subtilis agar plate to inoculate sterile NB
*(always study aseptically; disinfect bench surfaces, work close to flame and put the loops and needs etc over flame to remove any potential contamination)

Exercise 04*

1. Serial Dilutions: Make serial dilutions by using saline solution (0,85 %NaCl) up to 10-5 , 10-6 and 10-7 as described below:

And use following chart for each group;
Dilution Group #
——————–
10-5 1, 2, 3
10-6 4, 5, 6
10-7 7, 8

2. As a stock solution, use S. morcencens, while making solutions; be careful about mixing of solution.
3. After serial dilutions spread 0.1 ml of solution to plate and label together with dilution factor.
4. For pour plate, prepare hot and sterile NA and after cooling of agar mix 0.1ml of culture solution and pour directly to the plate.
5. Pour 0,1ml of diluted sample (in our group10-7 dilution rate) to plate by using spreader or rod. Firstly, flame spreader together with alcohol and cool it on the inner surface of Petri plate cover part, then spread sample gently and evenly.
6. To obtain discrete colonies from any kind of media such as NB or NA, take sample of bacteria by inoculum loop and by using “four corner method”. Firstly, sterilize loop over flame & cool it by waiting 10 sec, take sample from mixed culture tube and make parallel movements over agar, after that flame and sterilize loop again and take one line from this parallel lines by making one perpendicular line and make new & unrelated parallel lines and continue like this for two more time.
*(do not forget to label plates correctly, write name of group, date, dilution, species name, spread volume and kind of plating technique whether spread or pour)

FINALLY INCUBATE ALL INOCULATED CULTURES, PLATES AND TRANSFERS AT 370C FOR 24 HOURS AND RECORD YOUR FINDINGS.

Results:
A. Microorganisms in the Environment:
Time of exposure to air
#of colonies grown
0’
0
5’
5
10’
9
15’
15
20’
13
Source of Bacteria
#of colonies grown
Touch the surface of the agar with your finger
37
Combine your hair over agar
14
Cough onto agar 1 or 2 times
29

B. Broth Transfer: Some colony formation was obvious when we look at cloudy appearance.
C. Agar Slants as a Source of Inoculum: Some colony formation was obvious when we look at cloudy appearance.
D. Inoculation of an Agar Slant: There are many colonies formed in the zigzag form on the surface.
E. Transfer to Agar Deeps: Inside of the hole which is opened by needle contained many cells near the surface.
F. Colony Selection: Some colony formation was obvious when we look at cloudy appearance.

Exercise 4:
Group #
Dilution Rate
Colony # (pour)
Colony # (spread)
1
10-5
0
UC
2
10-5
0
UC
3
10-5
0
972
4
10-6
397
114
5
10-6
400
UC
6
10-6
UC
479
7
10-7
0
16
8
10-7
0
UC

(Colony numbers over 300 should be considered as uncountable)

Calculations:

Cfu/ml: #of colonies on plate x (1/dilution of solution)x(1/volume of inoculation)
Acceptable results:
10-5à972 (1) cfu/ml=972*(1/10-5)*(1/0,1ml)=9,72*108
10-6à114 (2) cfu/ml=114*(1/10-6)*(1/0,1ml)=1,14*109
10-7à16 (3) cfu/ml=16 *(1/10-7)*(1/0,1ml)= 1,6*109

cfu/ml Ratios:
(1)/(2)= 0,85 (1 is expected)
(2)/(3)= 0,71 (1 is expected)
(1)/(3)= 0,61 (1 is expected)

Colony # Ratios:
(1)/(2)= 8,5 (10 is expected)
(2)/(3)= 7,1 (10 is expected)
(1)/(3)= 60,8 (100 is expected)

Discussion:
In this experiment, we have learned several basic molecular biology techniques and use of equipments such as plating techniques (spread, streak, and pour).

Organisms live everywhere, especially microorganisms found in everyplace. In our experiments we showed that they are found in air and if you contact your nutrient media with air more and more you will get more number of colonies in general if conditions wind and place of nutrient media is similar or same. This is shown by up to 15 min, there were an increase in the number of colony but at 20 min there is a decrease in number. I think this is due to the place of plate remained opened and due to less circulation of air around open nutrient media. Moreover, I also can say simple although just one experiment is not enough finger touching can cause formation of more bacteria then coughing over it and combining of hair gives least number of colony, it means that our friend has clear hairs than any fingers and coughing. In addition to that it is necessary to note that it is normal to see bacteria colony formation from finger, hair because they are always contact with environment.
Because Nutrient Broths have all necessary nutrients necessary for grooving of any kind of bacteria in general, we observed growing of colonies in all NB tubes. Although we expected that in the “deep agar” tube bacteria are grown at the bottom, whereas in the “slant” tube bacteria are grown on the surface. For agar deeps, as indicated in the result part we could see bacterial formation around and inside of the hole formed by needle. Moreover, that is because S.aereus is an aerobic bacterium and tends to go to the surface of the agar and there was red pigmentation specific to S.aereus. In the case of inoculation of agar slats by zigzag form, we observed zigzag formation of colony growth after 24 hours at 370C incubation.
What about the serial dilutions and results? They are not successful in general, we could use just 3 results to calculate cfu/ml values. Moreover, results are not satisfying ten times decrease of colony number in the case of ten times dilution, in other words, ten times more colony formation in the previous dilution when we consider 16 colonies of 10-7 and 114 colonies of 10-6; ratio is 8,5. Reasons are, I suggest, improper dilutions, burst of cells in the case of strong wortexing although it is very difficult to kill bacterial cells by broken their cell wall. Actually, these unexpected results are expected if you are a new scientist working on a new subject. It is always possible to make errors; you should get experience by time with your failures.
In our group and in many groups we could not obverse any colony formation in the pour plates but in some groups we could observe characteristic colonies which is red colored colonies of.
During the dilutions we should use an isotonic solution like 0.85% saline solution to keep cells alive by preventing bursting due to osmolarity.
One biologist or geneticist may be I should also add microbiologist should know the importance of leaving of Petri plates inverted after spreading on to agar plates in to the incubator. If they are not left inverted, the water vaporizes from the agar and condenses on the lid and falls back. That causes increase of the dilution of bacteria on agar and also loss of water from agar so form more stringent conditions to live in.
We made three different types of plating techniwues; spread, streak, and pour. Spread plate was a general approach to study pure culture, isolating of pure culture. On the othe hand, streak plate was good to obtaining single colonies from any kind of stock like frozen or colony formed before. Pour plate was a good method to study bacteria which can live in an oxygen deficient environment and it was also easy to pour on just Petri plates together with proper mixing of desired bacteria. In our experiments, unfortunately, we failed to see colony formations neither on surface nor inside of NA. This may be due to killing of bacteria during addition to NA media, media might be very hot.
As a final discussion, there were small colony formations and pink colored big colony formations in NA because we used mix culture for this purpose.

I think no need to mention aseptic techniques that should be follow by researcher. I can not imagine plating without studying close to fire or inside of laminar flow to prevent contamination and use of loop without flame sterilization until giving red color.

In conclusion, these exercises were successful and they were a good start to study and learn more basic microbiology methods, because they are essential of microbiology studies. New procedures were practiced, and further understanding of transfers, plating techniques, logic of serial dilutions and cfu/ml calculations were gained.

REFERENCES:

1. Lab course notes….
2. Lab manual of microbiology, (bio355), Exercise 3&4. 

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