TRANSFER OF BACTERIA USING ASEPTIC TECHNIQUE

GENERAL GUIDELINES:
Safety Wear a lab coat and have your goggles on! ALWAYS disinfect the tables BEFORE and AFTER lab. Wash your hands with soap both BEFORE and AFTER lab, and, in addition, when you have a spill. Be sure to have nonessential materials (the ONLY essential thing is the lab handout and a notebook to write in) off of the table. Place test tubes in racks when working at your table: never lay the tubes down--they leak. Do not dump ANY microbial suspension down the drain--only in the discard area. The gas should be turned all of the way on, so that the level is parallel with the rubber tubing. THE LAST PERSON ON THE TABLE CHECKS THAT ALL 4 GAS JETS ARE OFF! If there is a spill: o Tell your instructor about it. o Flood the area of the spill with disinfectant and leave on for 10 minutes before using paper towels to soak up. Beginning the lab Label all test tubes and petri plates with your name (initials), your table #, date, exercise #, and name of organism. You always pick up your microorganisms as a set for your table, sharing them between the table members, and REPLACE THEM UNDER THE BIOSAFETY HOOD. Use masking tape only to mark on the tubes: You can use tape or mark directly on the agar plates. Technique ALWAYS use the proper aseptic technique when transferring cultures from one medium to another. Keep test tube caps and petri dish covers on media to reduce contamination (matters not whether it is sterile media or already cultured). Check for contamination in or on media. Remove contaminated media and place in discard area. Finishing the lab When using microbial cultures, take one of each organism for the table, use it, and then DISCARD properly. o Tubes in test tube rack discard o Agar plate in autoclave bag REMOVE ALL labels from test tubes when discarding. Be sure to put any unused media tubes BACK into the racks if unused: Replace unused agar plates back into bags if not used.

By now you know what media is: it is the nutrient-rich material that provides food to the microbes. There are 3 forms of media: An agar medium contains agar (1.5-2%) as a solidifying agent for isolation and purification. A broth medium has no agar: it is for fast, luxuriant growth. A semi-solid has a reduced percentage of agar, and, therefore, has qualities of both agar and broth. Fall 2011 - Jackie Reynolds, Richland College
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In addition to nutrients and growth factors, and perhaps agar, there are additives such as NaCl salt, pH buffers, and pH indicators that allow biochemical reactions to be identified. In this exercise you will learn how to subculture bacteria, using a variety of culture media as your inocula sources and as your new culture media. Different species of bacteria will be used so that you can become familiar with different growth patterns. You will also have a mixed culture with 2 species in order to learn how to best separate and isolate bacterial species. Streak plates are a great technique for separating mixed cultures into visibly separate species, which can then be isolated and grown as pure cultures on fresh media. Each colony represents a different clone of cells, each originating as a single mother cell. All agar plates are incubated UPSIDE DOWN (exceptions will be pointed out occasionally). WHY? It reduces bacterial contamination since the bacteria, even if they get into the plate in between the lid and bottom, would have to go UP to get to the agar. It reduces the possibility of water condensation that may be on the lid dropping onto the agar, causing fluid to run across the agar medium. ALWAYS check agar plates carefully to make sure that there are no mold or bacterial contaminants on the plate: if so, discard the plate in the autoclave bag. Do the same with any tube media that you pick up. If contaminated, discard the tube. or plate If you see water running on the agar plate, you can do 2 things: Place the agar plate upside down in the 37C incubator with the top cracked. Get another agar plate. OBJECTIVES: Subculture bacteria in/on sterile media of various forms. Eliminate potential contamination of bacterial cultures by using aseptic technique. Practice hand coordination required in good transfer techniques. Identify different ways by which bacteria grow in culturein agar deeps, on agar slants, on agar plates, in broths.

MATERIALS NEEDED: set of cultures for the table: a TSA (trypticase soy agar) slant culture of Bacillus subtilus a TSB (trypticase soy broth) culture of Staph epidermidis a TSA plate of E. coli sterile media: (per person) 2 TSB 1 TSA slant 1 TSA plate

THE PROCEDURES: BE SURE TO READ OVER THE STEP-BY-STEP DESCRIPTION OF TRANSFER TECHNIQUE BELOW BEFORE PERFORMING THE TRANSFERS. THE TRANSFERS: (performed individually) 1ST session: 1. Subculture a broth culture of Staph epidermidis into a TSB.. 2. Subculture a slant culture of Bacillus subtilus into TSB. 3. Subculture a plate culture of E. coli onto a TSA slant and onto a TSA plate. 4. Incubate all newly-made cultures at 25 or 37 degrees C, wherever your instructor directs you. 2nd session: 1. Check each culture for the presence of bacterial growth. 2. Use the interpretation section at the end to determine the bacteriums growth characteristics on a slant, in broth, and in deeps. Check the agar plate culture for growth. ASEPTIC TECHNIQUE: 1. Have both the culture that you are taking the inoculum from and the new, sterile medium in front of you. Be sure that the new medium is already labeled so you do not confuse the various cultures. Be sure that you have all inoculating equipment handy. 2. Pick up both tubes in the hand not using the inoculation instrument. 3. Heat the inoculating wire of the loop or needle until red-hot, and be sure that the ENTIRE wire is sterilized. You are now ready to pick the inoculum from the bacterial culture. Be sure to COOL your inoculation instrument a few seconds before picking the inoculum. If you hear a sizzle as it touches the medium, it was TOO HOT. Use an inoculating needle for agar deeps and an inoculating loop for the agar plate and the broths. 4. Keeping the sterile inoculation instrument in your hand, remove both tube caps with your little finger. 5. Run the tops of the tubes through the heat to create an updraft (taking air contaminants AWAY from the tube entrance). 6. Holding the tubes at a 45 degree angle, remove the inoculum and QUICKLY place the inoculum into the new medium tube. 7. Sterilize the tops of the tubes again (to eliminate potential air contamination again) and replace the caps. 8. Heat the inoculating wire of the loop or needle again before placing on the table. 9. Incubate the plates and tubes on the 25 degree C shelf. (room temperature) Look at the section below in INTERPRETION to read your tube results. TAKING THE INOCULUM --FROM A BROTH CULTURE: The inoculum is obtained by first shaking the culture a bit, and then going into the broth with the loop. A film of broth culture can be seen across the loop as you remove it from the tube.
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--FROM AN AGAR SLANT CULTURE: The inoculum is picked off of the top of the slant, like a scraping motion. --FROM AN AGAR PLATE CULTURE: The plate cultures have isolated colonies of bacteria growing on them. Pick only one colony or a bit of a colony, if big, with your loop. Lift the lid of the plate just a bit, in order to get your inoculating loop into it: DO NOT TAKE THE ENTIRE LID OFF. INOCULATING THE NEW MEDIUM --INTO A BROTH: The inoculum is just knocked around in the broth, and against the sides of the tube in the broth. --ONTO AN AGAR SLANT: Place the loop with the bacteria into the slant tube, all the way down to the bottom of the slant. There are 2 ways to inoculate the slant: If your goal is to identify the type of growth pattern, then just bring the loop straight up the slant. If your goal is to have a luxuriant culture, inoculate in a zig-zag pattern, starting at the bottom of the slant. This increases the surface area of the culture. --INTO AN AGAR DEEP: Use the needle to inoculate the deeps or semi-solid agars. Stab the inoculum down to the bottom of the deep in a clean, straight stroke. --ONTO AN AGAR PLATE:

1. In the pure culture technique exercise, you will learn how to make a streak plate but for right now just inoculate the agar plate with a zig-zag motion from top to bottom of the plate. 2. While doing this, lift the lid just enough to insert the loop underneath: this will reduce contamination. 3. When streaking the agar, keep the loop horizontal and only streak the surface of the agar: DO NOT DIG INTO THE AGAR. 4. Replace the lid and invert the plate. Incubate the plate.
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INTERPRETATION OF RESULTS: AFTER INCUBATION, check the growth patterns of all tubes and plates.

LABORATORY REPORT SHEET QUESTIONS:

1. Why streak from the bottom of the agar slant medium up to the top in a straight line, rather than a back and forth wavy inoculation from side-to-side on the slant?

2. Of what use is it to know what kind of growth pattern on an agar slant or a broth medium an organism has?

3. Why use an agar plate to grow a bacterium rather than an agar medium slant or a broth medium?

4. W h a t i s t h e p u r p o s e o f f l a m i n g t h e m o u t h o f t h e t u b e ?

5. How do you determine if an organism is growing in a broth medium?