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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 discardo 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 themicrobes. There are 3 forms of media:

An agar medium contains agar (1.5-2%) as a solidifying agent for isolation andpurification.

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 asNaCl 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 asyour inocula sources and as your new culture media. Different species of bacteria will beused so that you can become familiar with different growth patterns. You will also have amixed culture with 2 species in order to learn how to best separate and isolate bacterialspecies. Streak plates are a great technique for separating mixed cultures into visiblyseparate 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 bacterialcontaminants on the plate: if so, discard the plate in the autoclave bag. Do the same withany tube media that you pick up. I f contaminated, discard the tube. or plateIf 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 culture—in agar deeps, on agar slants, onagar plates, in broths.

MATERIALS NEEDED:

set of cultures for the table:

a TSA plate of E. colisterile media: (per person)

1 TSA slant1 TSA plate

a TSA (trypticase soy agar) slant culture of Bacillus subtilusa TSB (trypticase soy broth) culture of Staph epidermidis

2 TSB

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THE PROCEDURES:

BE SURE TO READ OVER THE STEP-BY-STEP DESCRIPTION OF TRANSFERTECHNIQUE BELOW BEFORE PERFORMING THE TRANSFERS.

THE TRANSFERS: (performed individually)1ST session:

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 bacterium’s 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 mediumin front of you. Be sure that the new medium is already labeled so you do not confuse thevarious 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, andbe sure that the ENTIRE wire is sterilized. You are now ready topick the inoculum from the bacterial culture.

Be sure to COOL your inoculation instrument a few secondsbefore picking the inoculum. If you hear a sizzle as it touchesthe medium, it was TOO HOT. Use an inoculating needle for agar deeps and an inoculatingloop for the agar plate and the broths.

4. Keeping the sterile inoculation instrument in your hand, removeboth tube caps with your little finger.5. Run the tops of the tubes through the heat to create an updraft (taking air contaminants AWAYfrom the tube entrance).6. Holding the tubes at a 45 degree angle, remove the inoculum and QUICKLY place theinoculum into the new medium tube.7. Sterilize the tops of the tubes again (to eliminate potential aircontamination again) and replace the caps.8. Heat the inoculating wire of the loop or needle again beforeplacing on the table. 9. Incubate the plates and tubes on the 25degree C shelf. (room temperature) Look at the section below inINTERPRETION 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 seenacross the loop as you remove it from the tube.

1. Subculture a broth culture of Staph epidermidis into a TSB..

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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 yourinoculating 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 thebroth.

--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 theslant.If your goal is to have a luxuriant culture, inoculate in a zig-zag pattern, starting at the bottomof 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 forright now just inoculate the agar plate with a zig-zag motion from top to bottom of theplate.

2. While doing this, lift the lid just enough to insert the loop underneath: this will reducecontamination.

3. When streaking the agar, keep the loop horizontal and only streak the surface of theagar: 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 brothmedium an organism has?

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

4. W hat i s t he pu rpose o f f lam ing the mou th o f the tube?

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