Isolation of Pure Cultures and Culture Storage Techniques

The objective of this experiment was to isolated a colony from mixed culture; and prepare long-term, mid-term and short-term culture storages.
Bacteria have some specific morphological features while growing. These specifity bacterial colonies have is observable by naked eye. Only interested bacterial colony can be taken from culture, a method called isolation. Scientists use this feature to achieve a culture that only a specific kind of bacteria live, it is called pure culture, and isolate it from environment.(1)
Mixed culture is the culture that at least two kinds of bacteria exist on plate. Mostly, pure culture is formed from a mixed culture. Scientists take a specific colony from mixed culture and then place onto pure culture by serving a media that only these interested bacteria can live there. Pure cultures are ideal to study on them.(1)
Colony is an apparent cluster of bacteria that grow from a single cell and living on solid media. Colony can include millions bacteria and this huge numbers cause that they are visible for naked eye. Also these bacteria are same genetically because they come from a single mother cell.(1)
Differences of colonies morphologically can be classified as size, shape, pigmentation, opacity, margin, texture, elevation. The most used morphological feature is texture. Colony can have circle borders between and media, it is called smooth colony, but some colonies have irregular border between and media, it is called rough colony.(1)
Storage of pure culture:
Bacterial colonies must be storage for a while to study on them. According to duration of studying, they can be storage in long-term, mid-term and short term. (2, 3 and 4)
Firstly, short-term is used for one-week researching. Bacterial solution is placed on solid media as streaks or spreading. After one-week incubation and generally 40C are used for short-term. (2, 3 and 4)
Secondly, mid-term is used for one-month researching. Semi-solid media is proper to mid-term storage. This semi-solid media is formed by slant agar, it is produced by solidifying. It can give a large surface area for bacteria can place. By time periods, bacteria can be placed into new media and time can be expended. (2, 3 and 4)
Thirdly, bacteria can be waited for years by long-term storage. Long-term storage is based on to get slow metabolism of cells. It is realized by taking away water from cells without hurting them. For long-term storage, some techniques are used and these are (2, 3 and 4):
⦁ Lyophilization( Freezing-drying): this technique is used that frozen water is heated rapidly and is vacuumed from cells. The technique can include differences on values or according to kind of microorganism. Basically, lyoportectant is added to help freezing by protecting cells. It can be skim milk, or a special glucose solution. After freezing, in a machine water is vapored directly and then cells are left without water immediately.
⦁ Liquid Nitrogen: Liquid nitrogen is used as freezer for cells. Liquid N is -1960C. This method needs a protective buffer like solutions including glycerol, it is called cyroprotectant. The temperature is reduced 10C by 10C. According to species, it is stopped at a point.
⦁ Glycerol: Frozen cells are kept at -800C in glycerol by protecting cyroprotectant. 40% glycerol can serve a protection against osmotic pressure.

Material and Method:
Mixed culture
Dishes with solid media
Tubes including semi-solid media
Bunsen burner
40% Glycerol
Slant agars
Eppendorf tubes
Liquid media

First of all, the tubes that have been numbered (13, 14, 15) differently was used for this experiment. Each tube has one of; E.coli, B.subtilis or mixed culture of both of them.

For Preparation of Short-term Media:
The bacterial colonies that have been prepared before on agar plates were observed and written visual features of colonies with naked eye to detect whether mixed colonies which bacteria exist in these colonies. Unknown colonies were taken and then placed onto our agar plates by being careful about aseptic protocols. After placing, dishes were labeled with experiment’s name, date, name of the sample. Next day, bacterial colonies taken from unknown colonies were investigated according to morphology of colonies. Colonies were detected mixed colony or that colonies belong to kind of bacteria (E.coli, B.subtilis).

For Preparation of Mid-term Media:
At mid-term media, 2 techniques were used: slant and stab culture.
⦁ Slant Culture:
Firstly, needle was hold in flame until it has a red color to be sterilized. After opening the tube that includes bacteria, the tube was passed through flame. The needle was put into the tube and then a snake-like line was formed from beginning point to end point of slope in tube. After that, the needle was submerged into slant agar. Lastly, the tube was labeled with date, experimenter’s name and number of the tube that includes pure culture of one of bacteria or mixed culture. Next day, the tubes were observed and detected kind of culture and bacteria the numbered tubes have.

⦁ Stab Culture: The needle was waited in flame until red color. The sample was taken from the opened tube after sterilization with flame and was put in agar by stabbing upright. After transferring, used bacterial tube was passed through flame and then closed. The tube with agar labelled with same like the other steps. Next day, tubes were observed.

At 2 mid-term culturing methods. The tube with agar was not closed tightly to provide air exchange for bacteria.

For Preparation of Long-term Media:
1 ml of 40% glycerol and 1 ml liquid media was mixed in a tube, and then incubated E.coli was transferred into the tube before mixing. The solution was poured into fresh and clean Eppendorf tubes. At all steps, the tube was kept on ice. This culturing was prepared for just seeing how to prepare long-term culture. Last step, keeping at -800C wasn’t realized.


Figure 1- Bacterial colony that grow on slant agar after slant culturing(Left), colony can be seen as snake-like lines ;and bacterial colony that grow on stab line in agar after stab culturing (Right), colony can be seen as a line in figure.

Figure 2- The samples that taken from the numbered tubes. The dish where 2 differnet colonies grow, mixed culture including E.coli and B.subtilis colonies that was taken from Tube 13 (Left). B.subtilis can be said on the dish that was transferred form Tube 14 according to morphology of colonies (Middle). E.coli colonies that were transferred from Tube 15 are on the agar. (Right)

Shape Margin Elevation Size Texture Appearance Pigmentation Opacity
E.coli circular entire flat small smooth dull no pigment opal
B.subtilis irregular undulate convex moderate rough dull no pigment opal
Tube 13 circular and irregular entire
and undulate flat and convex small and moderate smooth and rough dull no pigment opal
Tube 14 irregular undulate convex moderate rough dull no pigment opal
Tube 15 circular entire flat small smooth dull no pigment opal
Table 1- The morphology of colonies, first two samples was used for detecting and differing kinds of bacteria after culturing on dishes. The others show that morphology of bacterial colonies according to “number” of tube transferred (Tube 13, 14 and 15).

E.coli and B.subtilis colony morphologies was recorded (Table 1) and observations according to table were done after overnight. Tube 13 was detected that it includes mixed culture because of 2 different morphologies of colonies (Table 1) by comparing with the previous observation. Tube 14 was B.subtilis. The observations (Figure 2 and Table 1) was confirmed this. Tube 15 was determined that it includes E.coli by Figure 2 and Table 1. Also any contamination couldn’t be seen.
Slant and stab methods have bacterial culture. Some bacteria could grow at slant method better and some at stab method better.
Long-term storage was just prepared to learn how to prepare.

What is intended in this experiment is to detect kinds of bacteria by using morphological features of their colonies and form storage for them.
At first step the dishes including unknown bacterial culture, E.coli, B.subtilis or mixed culture of both of them were observed for detected what kinds of culture are by using morphological features. Mixed culture could be distinguishable easily because mixed culture has 2 different colony types. Firstly, size can be shown for difference. Also shape, margin, elevation and texture include differences. However some morphological features were quite similar. These are appearance, pigmentation, opacity.
For observing differences better, the other cultures were investigated. The first observable colonies observed in petri dishes were small. Also they had regular colonies. Circular spread of them can be seen. The surface between outside of a colony and the colony was so clear and regular, so margin of this colony was entire. Bacteria tended to expend to surface, flat. As an entire colony, they were smooth colonies. Observations were showing that these bacteria are E.coli. Second group bacteria should have been B.subtilis and morphological critics was confirming. On the other hand, both different colonies have mutual features like being non-pigmentation, dull and opal at appearance, pigmentation, and opacity. (Figure 2 and Table 1)
At mid-term storage, bacterial cultures were observed that they relatively grow well for both methods. Some colonies have spread better slant method. B.subtilis, a kind of bacteria can do aerobic respiration, could be thought. (Figure 1)
Long-term storage was prepared for only seeing preparation of long-term storage.
[1] Healthwise, Incorporated (2010-06-28). “Throat Culture”. WebMD
[2] Old, D.C.; Duguid, J.P. (1970). “Selective Outgrowth of Fimbriate Bacteria in Static Liquid Medium”. Journal of Bacteriology. American Society for Microbiology. 103 (2): 447–456.
[3] Madigan, Michael T. (2012). Brock biology of microorganisms (13th ed.). San Francisco: Benjamin Cummings
[4] Uruburu, F. (2003). “History and services of culture collections”. International Microbiology. 6 (2): 101–103.

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