ASEPTIC TRANSFER & PURE CULTURE TECHNIQUES

GENERAL GUIDELINES & REMINDERS:

SAFETY: NO EATING OR DRINKING IN THE 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 handouts and anotebook to write in) off of the table.

ALWAYS disinfect the tables BEFORE and AFTER lab. 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 anytubed media that you pick up: if contaminated, discard the tube in the discard racks.

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. 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 TO MAKE SURE THAT ALL 4 GAS JETSARE OFF!

INOCULATION, INCUBATION AND LABELING OF CULTURES: You always pick up your microorganisms as a set for your table, sharing them between

the table members, and replace them in the biosafety hood. Keep test tube caps and petri dish covers on media to reduce contamination (matters not

whether it is sterile media or already cultured). If you see water running on the agar plate, you can do 2 things:

o Place the agar plate upside down in the 37C incubator with the top cracked until dry..o Get another agar plate.

Place test tubes in racks when working at your table: never lay the tubes down—they leak. Heat the entire piece of metal of the inoculation instrument in the flame: it should be RED HOT.

Be sure to COOL your inoculation instrument before picking the inoculum. Use an inoculating needle for agar deeps and an inoculating loop for agar plates and broths. ALWAYS use the proper aseptic technique when transferring cultures from one medium to

another. Be sure to put any extra media tubes BACK into the racks if unused: Replace agar plates

back into bags if not used. Label all test tubes and petri plates with your name (initials), your table #, date, exercise #, and

name of organism. Use masking tape to mark on for the tubes: You can use tape or mark directly on the plates.

DISPOSAL OF CULTURES: Do not dump ANY microbial suspension down the drain—only on the discard cart. REMOVE ALL labels from test tubes when putting them on the discard cart. Tubes for disposal go inn test tube racks at disposal area in corner of the lab. Agar plates for disposal go into the red biohazard autoclave bag on the floor below the tubes.

All agar plates are incubated UPSIDE DOWN (exceptions will be pointed out occasionally).WHY?

1. It reduces bacterial contamination since the bacteria, even if they get into the plate inbetween the lid and bottom, would have to go UP to get to the agar.

2. It reduces the possibility of water condensation that may be on the lid dropping onto theagar, causing fluid to run across the agar medium.

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---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

types.

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.

THE 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.Streak out cultures for isolation and identify colonies.

MATERIALS NEEDED:set of cultures for the table:

a culture of E. coli and Staphylococcus mixed togethersterile media: (per person) 3 TSB

2 TSA slants2 TSA plates

THE PROCEDURES: per personThere are descriptions how to inoculate an agar plate, a broth tube, an agar slant, and so onbelow .

a TSA (trypticase soy agar) slant culture of Bacillus subtilisa TSB (trypticase soy broth) culture of Staph epidermidisa TSA plate culture of E. coli

THE TRANSFERS YOU WILL BE DOING IN LAB:

Subculture a broth culture of Staph epidermidis onto a TSA slantSubculture a slant culture of Bacillus subtilus into TSB.

Subculture a mix of E. coli and Staphylococcus in TSB and on a TSA plate

Although your instructor will show you how to perform these procedures,here are the methods you will use.

ASEPTIC TECHNIQUE:1. Have both the culture that you are taking the inoculumfrom and the new, sterile medium in front of you. Be surethat the new medium is already labeled so you do notconfuse the various cultures.2. Pick up both tubes in the hand not using the inoculationinstrument.

hot, and be sure that the ENTIRE wire is sterilized. You arenow ready to pick the inoculum from the bacterial culture.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 anupdraft (taking air contaminants AWAY from the tube entrance).6. Go into the tube to take your inoculum and QUICKLY place the inoculum into the newmedium tube.7. Sterilize the tops of the tubes again (to eliminate potential air contamination again) andreplace the caps.8. Incubate the plates and tubes in the 30 degree C incubator. Look at the section below inINTERPRETION to read your tube results.

Subculture TSA plate culture of E. coli into TSB, a TSA slant, and a TSA plate.

Use an inoculating loop for the agar plate and the broth media.

3. Heat the inoculating wire of the loop until red-

TAKING THE INOCULUM:FROM A BROTH CULTURE:

The inoculum is obtained by first shaking the culture a bit, and then going into the brothwith the loop. A film of broth culture can be seen across the loop as you remove it from thetube.

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 toget your inoculating loop into it: DO NOT TAKE THE ENTIRE LID OFF.

INOCULATING THE NEW MEDIUMTO A BROTH CULTURE:

The inoculum is just knocked around in the broth, and against the sides of the tube in thebroth.

TO AN AGAR SLANT CULTURE: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 loopstraight up the slant.

If your goal is to have a luxuriant culture, inoculate in a zig-zag pattern, startingat the bottom of the slant. This increases the surface area of the culture.

TO AN AGAR PLATE CULTURE: see directions below on streak technique

TO 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.

STREAKING AN AGAR PLATE:Until you become well-acquainted with this procedure, you might want to draw the 3 sectionsthat you will streak inside of, on the back (bottom of plate containing agar medium) with asharpie pen. Keep in mind that you flame your inoculating loop between each sectionstreaked.

1. Pick up a loopful of your inoculum from either a broth or an agar culture. Using a sterileagar medium plate (lift the lid just enough to insert the loop), streak a vertical line straightdown.2. When streaking the agar, keep the loop horizontal and only streak the surface of the agar:DO NOT DIG INTO THE AGAR.3. Move the loop in a zig-zag pattern across the agar until 1/3 of the plate is covered, finishingthe first section.4. Sterilize the loop in the flame and let it cool before continuing to spread the bacteria. Youcan do this by 1) sticking the hot loop in the agar at the edge of the agar away from thebacteria, or 2) just holding the loop for a few seconds while it cools.5. Rotate the plate about 90 degrees and spread the bacteria from the first streak into asecond area using the same zig-zag spread technique.6. Sterilize the loop again. Rotate the plate about 90 degrees and spread the bacteria fromthe second streak into the 3rd area in the same pattern.7. Sterilize the loop again. Replace the lid and invert the plate. Incubate the plate.

INTERPRETATION OF RESULTS:AFTER INCUBATION, check the growth patterns of all tubes and plates.

GROWTH PATTERN ON AN ISOLATION STREAK PLATE

You should see bacterial cells growing along streak lines and in isolated areas.