PURE CULTURE TECHNIQUES

Most specimens (from animal tissue, plant tissue, or environmental samples) will be mixed,with a variety of bacteria (or other microorganisms). A single gram of feces, for example, hasover 1010 bacteria and that gram would have over 20 different bacterial species.

Particularly in a medical setting, where a patient’s body fluid or tissue (skin, blood, spinalfluid, urine) is sent to the microbiology lab for analysis, the specimens will most often bemixed. In order to identify the bacteria and run antibiotic susceptibilities on the causativeagent of the infection, the microbiologist must be working with a pure culture of the bacterium.

This exercise begins with a mixed culture of bacteria and will end with, hopefully, purecultures of the 2 bacteria. Luckily, the 2 bacteria being used look different from each otherwhen growing on agar plates. Two different types of agar plate isolation techniques will beused---streak and pour. Each method has advantages and disadvantages, and particularuses. The same mixed culture will be used for both methods. Another technique, called thespread plate, is used commonly for counts, as is the pour plate technique. It uses pre-madeagar plates, with the fluid inoculum being placed on top of the agar medium. The inoculum isthen spread around on the plate with a bent glass rod. That procedure will not be donetoday.

This also gives you a chance to see how agar plates are made. Some of the TSA plates arealready made: others will be made by you. This requires that liquefied agar medium (sittingin a water bath) be kept above the solidification temperature. Agar’s solidificationtemperature is 42 degrees C, but its liquefaction temperature is 100 degrees C. Whensterilized in an autoclave or boiled, the medium will stay liquid until the temperature gets to 42C. When that happens, the medium will solidify very fast. If it solidifies on you before youfinish making your cultures, it has to be disposed of (if not used yet, it can be placed back inthe sterile media racks).

REMEMBERALWAYS check agar plates carefully to make sure that there are no mold or bacterialcontaminants on the plate: i f so, discard the plate in the autoclave bag.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:

Compare different agar plate isolation techniques.Differentiate among various colony morphologies.

Fall 2011 – Jackie Reynolds, Richland College

2

MATERIALS NEEDED:

water bath at 50 degrees Cper person: 1 TSA plate

3 TSA plates

THE PROCEDURES:

Each student will make 1 streak plate from the mix, incubated at 25 CEach table will make a set of 3 pour plates from the mix, incubated at 25 C; an extra streakplate from the mix, incubated at 37 C; AND 2 streak plates during the 2nd session.

POUR PLATE TECHNIQUE (performed by the table)

You will dilute the bacterial sample by transferring loopfuls of the specimen from media tubeto media tube, with fewer bacteria ending up in successive tubes. The solidified agar deeps

After adding the bacterial samples, the agar is poured into sterile petri dishes and incubated.

on the bottom of the dish.3. Remove 1 tube of liquefied TSA from the water bath and take it to your table. DO

4. Pick up a loopful of your inoculum from the mixed bacterial culture and transfer itASEPTICALLY into the first tube of liquefied TSA.

5. Mix this tube #1 by rotating it quickly between your 2 hands: If done adequately, therewill be good mixing of the bacteria into the medium.. DO NOT invert the tube of agar.

6. From tube #1, aseptically transfer a loopful of the medium aseptically into tube #2.7. BE SURE THAT YOUR PETRI DISH IS SITTING RIGHT-SIDE UP. Pour the entire

contents of tube #1 into the Petri dish marked #1. Open the larger top of the dishRIGHT BEFORE pouring the agar into the dish, and replace the top as soon as

have been boiled and liquefied (agar medium will liquefy at 100 C), then cooled in a water bath.Without cooling the hot agar medium down, your bacterial specimens would be cooked..

UNTIL you are ready to put your bacterial specimens into the agar tubes,2. Obtain sterile Petri dishes (make sure that cover stays on) and label as #1, #2, and #3

1. Find the water baths containing the warm agar media. Leave the tubes in the bath

per table: 3 TSA agar deeps, liquified (in water bath)

NOT carry all 3 tubes at once: the others will solidify before you can inoculate them.

a culture of E. coli and another bacterium (preferably Serratia, or Staph) mixed together in TSB

plate-sized circle to spread the agar-bacterial specimen out well in the agar plate.finished. Do this quickly. Gently swirl the closed plate about 4-5 times in a large dinner

3 sterile Petri dishes (into which agar medium will be poured)

3

8. Mix tube #2. From this tube, aseptically transfer a loopful of the medium asepticallyinto tube #3. Mix the tube well by rotating it between your hands quickly.

9. Pour the entire contents of tube #2 into the bottom of the Petri dish marked #2. Gentlyswirl the closed plate in large circles o spread the bacterial out well in the agar.

10.Mix tube #3. Pour the entire contents of tube #3 into the bottom of the Petri dishmarked #3. Gently swirl the closed plate in large circles to spread the bacterial outwell in the agar.

11. Let the 3 plates solidify for at least 15 minutes before incubating. Invert the agarplates when placing them at 25 C.

12. After incubation, check the 3 pour plates for colonies and their features in/on theagar.

STREAK PLATE TECHNIQUE: (performed individually and as a table)Subculture a mix of E. coli and Serratia marcescens on a TSA plate, using 2 different streaktechniques

1ST session: each student streaks a plate from the mix, and the table does an extra plate

In this first technique, you will flame the loop between each plate section, thereby diluting thesample as you move from section to section. Remember that you go into the mixed culturetube ONLY ONCE, before the first section.

1. Until you become well-acquaintedwith this procedure, you might wantto draw the 3 sections that you willstreak inside of, on the back (bottomof plate containing agar medium)with a sharpie pen.

2. Pick up a loopful of your inoculumfrom the mixed bacterial culture.Using a sterile agar medium plate,streak a vertical line straight down. While doing this, lift the lid just enough to insertthe loop underneath: this will reduce contamination.

When streaking the agar, keep the loop horizontal and only streak the surface of theagar: 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,finishing the first section. Remember to close the lid on the Petri dish betweenstreakings.

4. Sterilize the loop in the flame and let it cool before continuing to spread the bacteria.You can do this by 1) sticking the hot loop in the agar at the edge of the agar awayfrom the bacteria, 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. Lift the lid only enough toeffectively streak.

6. Sterilize the loop again. Rotate the plate about 90 degrees and spread the bacteriafrom the second streak into the 3rd area in the same pattern. Replace the lid.

7. Sterilize the loop again. Invert the plate. Incubate the plate at 25 C.8. Each table will also make an extra streak plate from the mix, incubating it at 37 C. This plate

4

will be compared to one incubated at 25 C.

2nd session:1. Check the agar plate culture of the mixed culture for 2 very different colony types. E.

coli colonies will be rather transparent and large. Serratia marcescens colonies will bepink/orange, about the same size as E. coli. Also, check your streak technique or getyour instructor to do so, for feedback.

2. Your table will make 2 more streak plates to produce 2 pure cultures of the E. coliand Serratia marcescens. Stay away from merging colonies or close colonies.Subculturing in that situation will increase the chances of picking up another mixedculture, consisting of the 2 species that were merged together. ALWAYS pick a well-isolated colony when subculturing.

a. Pick a colony of each of the 2 different species of bacteria onto 2 new TSA agarplates, using your best aseptic streak technique.

b. Incubate at 25 degrees C.c. Check the plates for purity the next period.

INTERPRETATION OF RESULTS:

1. AFTER INCUBATION, check the growth patterns of all plates.Compare the size, shape, and location of colonies on the 3 kinds of plates.

Where are the colonies in the pour plates? Why?Why is there so much variation in colony sizes and shapes in the pour plates?Which method is best for accurate counting?Which method is the easiest to do?Which method gives easy access to the colonies---for subculturing to another

medium? Which method has the greatest space between the colonies?

2. Compare the streak plates incubated at 25 C and 37 C. Check pigmentation.3. After the 2nd session, where you made 2 streak plates, check those plates to make surethat you have 2 pure cultures of the 2 bacteria.

5

LABORATORY REPORT SHEET

QUESTIONS:

1. When performing the streak technique, why do you cross over back the 2nd streaksection back into the 1st section, and from the 3rd section back across the 2ndsection?

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

3. What is the purpose of flaming the mouth of the tube?

4. On the agar plate culture of E. coli and Serratia marcescens mixed together, were thecolonies in the first section of the plate the same size as those in the 3rd section?

Explain why this would happen.

5. Why are the colonies within the agar of a pour plate smaller than those on the surfaceof the agar?

6. What difference was seen in the Serratia colonies grown at 25 C and 37 C? Why?

7. What procedure has been used to make this culture plate---pour, spread, or streak?

Does it have good isolation? Why?