University of Bahrain College of Science Department of biology

BIOLS 465 {Gene Technology}

Lab # 2 Bacterial Cultural Techniques

Name: Bisma Bashir Mohammad ID # 20094704 Date: March 7, 2012

Aim
To study different techniques for culturing bacteria such as streaking, overnight suspension culture and midlog phase of E.coli culture.

Introduction
To culture bacteria, aseptic techniques are required. These techniques include heating instruments over Bunsen burner, flaming necks of the tube and opening lid of petri dish as little as possible. These techniques avoid contamination of cultures from air-borne microbes. To follow the growth of bacteria, different methods are used, for e.g. Streaking: streaking is a qualitative and rapid method to culture bacteria. Also isolated colonies can be obtained during streaking. Antibiotics and plasmid-borne resistance to antibiotics can also be isolated. Antibiotic resistance bacteria can grow in a media containing that Fig. 1 Action of antibiotics on cell wall of bacteria antibiotic. Antibiotics work either bactericidal or bacteriostatic. Bactericidal antibiotics kill the microorganism by preventing the production of cell wall material i.e. peptidoglycan due to which they continue growing without dividing or developing new cell wall. Therefore the cell gets weaker and eventually ruptures. On the other hand, bacteriostatic antibiotics inhibit the growth of microbes by binding to ribosomes; this means the cell cannot make proteins and therefore can't grow [1]. To speed up the process of growth in a lab, overnight suspension cultures are used. Because E.coli used in this lab has simple nutritional requirements and grow relatively slow on minimal media which has glucose as an energy source, salts, vitamin biotin and nucleoside thymidine. So it is preferred to use an overnight suspension culture which has passed its lag phase. Using that overnight suspension culture, which has passed its lag phase, plasmid can be inserted in it as it has become competant to uptake that desired plasmid because the ration of mid-log cells to competant cells is 10:1 [2]. The growth curve of E.coli is divided into 4 phases.
Fig. 2 Growth curve of bacteria

a. Lag phase in which slow growth occurs. Here bacteria adapt to new enviornment. b. Log phase where all metabolic reactions occur fast so bacteria grows exponntially. c. Stationary phase in which cell growth equals cell death. d. Decline/Death phase where all the nutrients in the media are used up and death overtakes [3].

Materials and Methods

Method 1: For streaking, 2 plates were used which were plain LB agar and other 2 were LB/amp i.e. had ampicillin in it. For each LB plates, one was streaked with culture without plasmid and other with culture with plasmid. Similarly, for each LB/amp plates, one was streaked with culture without plasmid and other with culture with plasmid. During these processes aseptic technique was adopted to avoid any contamination [2]. Method 2: An overnight suspension culture was prepared from a stock culture of bacteria by taking a loop full of culture and inoculating in a fresh LB broth media and then incubating the culture for 12-24 hours at 37C with continuously shaking [2]. Method 3: To get the mid-log phase of E.coli, overnight culture prepared in method 2 was used. 2.5mL of the suspension was transferred into 250mL of LB broth at room temperature. Again aseptic techniques were used in every step to ensure and contamination. Its absorbance was measure using spectrophotometer at 600nm and then after approx. 50min. its absorbance was measured again to check the growth. Then after every 20min. its absorbance was measured and average of the class was taken to plot against time to obtain a growth curve. Spectrophotometer was blanked with plain LB broth before transferring the overnight culture in LB broth [2].

Results
Streaking: -pAMP +pAMP KEY +ve = growth (colonies formed) +ve MM294 -ve MM294 -ve = no growth (no colonies)

+ve MM294 pAMP

+ve MM294 pAMP

Mid-log phase of E.coli growth culture. Time in min. 0 50 70 90 110

1 0

Group 5 Absorbance 0.045 0.045 0.061 0.086 0.141

Class Absorbance Average 0.0430 0.0430 0.0640 0.1232 0.2010

Growth Culture for E.coli strain MM294

20 40 60 80 100 120

Absorbance (O.D) at 600nm

Log phase
0.1

Lag phase

0.01

Time (minutes)

Discussion
Results explain us that plasmid containing ampicillin resistant gene can grow in an ampicillin medium as it has the ability to break the -lactam ring in ampicillin whereas normal strain cant grow as it has no gene for ampicillin resistance. Plain LB agar grew both the normal and plasmid containing strain as it has no ampicillin to interfere with the peptidoglycan synthesis. These were the expected results. Kanamycin, another antibiotic interferes with protein synthesis and does not allow both the strains to grow as they dont have Kanamycin resistant gene. The only resistant gene present is for ampicillin. Streaking was done in a zigzag pattern to get isolate colonies which can be then used to grow large cultures for the plasmid. In zigzag pattern, new streak was made to intersect the previous one at only single point so that only few cells are moved along and they can grow as single colonies. In overnight suspension culture, lag phase has been passed and cells have entered the log phase in which cells are dividing rapidly. This is important as these cells have become competent for the uptake of plasmid. Also the culture must be continuously shaken to ensure optimum growth as it provides air, exchanges nutrients and also flushes away the metabolic wastes.

Mid-log phase used, are from the overnight cultures. To reach the mid-log phase the cultures were transferred to fresh LB broth to get more nutrients and to speed up the growth. Here again the cells pass through a short lag phase and then grow rapidly to the mid-log phase. This can be influenced by the stage of inoculum, nutrients present in the fresh Lb broth and the temperature and pH of the media. If instead of mid-log culture, growth was started with the colony, it would have taken log time. Graph between the average absorbances of the class result v/s the time was plotted which showed the lag and log phases. It didnt reach the stationary phase so we cant find its mid-log phase which is required for further plasmid insertion. In our lab, results were delayed because a different strain i.e. E.coli K12 was used instead of E.coli MM294. This happened at the last moment and new culture could not be made so different strain was used. This strain has a different generation time and grows more slowly than that of MM249. Also heating the pipette before inoculating could delay the process. Another reason of the delay could be that the initial inoculum was not accurately taken.

Conclusion
We have successfully studied few bacterial culture techniques which can help to grow large cultures of plasmid. Also which method can be used to speed up the growth process. Advantage of using mid-log phase has also been studied. We also studied from our mistake that different strains of the same bacteria have different generation time

Literature Cited
1. Tortora, G. J.; Funke, B. R., Case, C. L. (2007): Microbiology: An Introduction, 9 th edition, The Benjamin/Cummings Publishing Company Inc., California. 2. Bloom, Freyer, Micklos (1995): Laboratory 2 in Laboratory DNA Science, Benjamin/Cummings Publishing Company Inc., California. 3. Stephen Claydon (2006-2008): Bacteria. Accessed: March 16, 2012. Available: http://scienceaid.co.uk/biology/micro/bacteria.html