LAB 5 (Data sheet 1.1)
MEDIA PREPARATION AND PLATE EXPOSURE
Culture Media Classification
1. By Consistency
2. By Contents
- All-purpose
- Selective
- Enriched
Consistency refers to a liquid, solid, or semi-solid. The use of a semi-solid allows us to do two things: see if the microbe is motile (can move), and to see if the microbe is aerobic or anaerobic.
All Purpose media
Nutrient agar (NA) does not support fastidious organisms, so NA is a good all-purpose media. It will support the growth of a wide variety of organisms. It is also inexpensive.
Selective media
This type of media selects for a particular type of organism to grow.
An example is Sabouraud’s Dextrose Agar (SDA), which has a high sugar content and an acidic pH. SDA isolates molds and yeasts, which do well in high sugar content, whereas bacteria are inhibited. Therefore, sugar acts as a bacterial preservative; that’s why jams, jellies, and preserves don’t get bacteria growth. However, molds are aerobic and they like sugar. They can get into your jelly jar when their microscopic air-borne spores drift in whenever the jelly jar is opened. Toss out the whole jar! Molds and yeast also grow well in an acidic environment.
SDA was originally designed to isolate molds of skin, nails, and hair, called dermatophytes, which are opportunistic pathogens. Dermatophytes produce ringworm, especially athlete’s foot. Yeasts are known for causing infections in women, diabetic and cancer patients. Since people with diabetes have a high sugar content, they are susceptible to such infections. Scrape off a little skin and place in SDA to isolate the microbe. SDA has 40 grams of glucose and a pH of 5.6.
Enriched Media
This type of media has added nutrients such as vitamins and amino acids. It is used to grow bacteria which are hard to grow, known as FASTIDIOUS organisms. Many pathogens are fastidious. An example of enriched media is Blood Agar, which has Trypic Soy Agar (made from soy) and 5% sheep’s blood (defibrinated to keep it from clotting).
PREPARATION OF SDA AND NA PLATES
The SDA and NA have already been prepared and sterilized in the autoclave by the previous lab group. The flasks are in the water bath at 50° C.
Why do we keep the agar at 50° C?
1. If it was 60° C it would be too hot to handle, plus it would take longer to cool. This will cause additional condensation on the inside of the lid as it cools.
2. If it was any cooler, it would harden before we could pour it. It hardens at 42° C.
Each person will get a gas-sterilized empty Petri dish.
Each lab table will get out one Bunsen burner.
Half of the class will pour NA plates and the other half will pour SDA plates.
We need to get the molten (melted) agar into the Petri dishes by using aseptic technique.
Aseptic technique
1. Take the stopper off the flask with your non-dominant hand.
2. Pick up the flask with your dominant hand and flame the edge of the flask.
3. Lift the lid off the Petri dish with the hand that is holding the sponge stopper. Keep the lid over the bottom of the plate so microbes don’t sink in from the air.
4. Pour enough agar into the dish to just fill the bottom. Make sure you don’t touch the flask to the Petri dish, or you will get contamination. The only other way to reduce contamination is to work under a hood with a UV light to kill bacteria.
5. Flame the flask opening again and put the stopper back on. Give the flask to the next person.
6. While the Petri dish is cooling, allow it to vent for about one minute by keeping the cover just a little bit open.
7. After you have poured it, do not touch it again. Let it sit until it has solidified (it will turn from clear to opaque). When it is solid, replace the cover and flip the plate over. Warm air touching the cool desktop will cause condensation to form inside the lid. This condensation is sterile, so it won’t contaminate the media. However, we want the surface of the media to be dry. Plates always need to be stored upside down.
8. Label the bottom of the plate with the type of agar that was used.
Preparation of new media for the next lab
Half the class will make 250 ml of NA per lab table, and the other half will make 250 ml SDA per lab table.
SDA: The jar says to use 65 grams per liter. We are making 250 ml (1/4 of a liter). How do we calculate how much SDA powder to use? 65 divided by 4 = 16.25 grams.
NA: The jar says to use 23 grams per liter. We are making 250 ml. How much NA powder do we use? 23 divided by 4 = 5.75 grams.
In both cases, proceed as follows:
There are only four weighing scales, so share them. Plug them in, and make sure they are set for grams and NOT ounces! Place the plastic weighing boat on the scale and press “tare” to make it go to zero. Use the metal spatula to add the number of grams required. Fold the weighing boat diagonally and pour it into a 500ml flask. Get a graduated cylinder with exactly 250 ml water in it, and pour a little water into the plastic weighing boat. Swirl to catch the leftover powder, then pout into the flask. Repeat this three times. Then pour the rest of the water into the flask.
Add a stir bar to the flask. Set the flask on a heating pad turned all the way up. KEEP AN EYE ON THE FLASK AND WAIT UNTIL THE MOMENT IT STARTS TO BOIL. Then remove it from the heat using an oven mit. If the flask boils for two long it will foam up and over, and you will lose the media.
Remove the stir bar with a magnet. Put a foam sponge on as a stopper.
Label the front of the flask with a grease pencil “NA” or “SDA”, and take to the autoclave room.
Place the flasks in buckets and set in the autoclave. Make sure it is set for “Cycle 10”, which is 20 minutes @ 121° C. Press the foot lever to close the door, then press the start button. In one hour, it will be done; press the foot lever to open door. Remove flasks and put in water bath.
Broths and Slants
Broths are the same nutrients as NA, but no agar. They do not have to be boiled, just stirred. They are placed in tubes instead of flasks and autoclaved as usual.
Slants are made exactly as NA, except they are poured into tubes instead of plates. After they are removed from the autoclave, they are placed on slant racks to cool so the agar in the tube stays at a slant.
Why use a slant?
We can inoculate just the top of a slant to get growth of aerobic bacteria, or we can stab a needle of bacteria into the tube to see if there are any microbes that can grow without air (anaerobic). Also, Petri dishes will only keep fresh for 3 weeks and then they dry out. Tubes will last for a long time in the refrigerator, and are useful for making stock cultures (pure cultures).
INNOCULATE YOUR PLATES
Make sure your poured plates are labeled on the bottom as SDA or NA. Also put your name on the plate bottom, and write “air exposure”. Remove the top and take it with you on a 15-minute break. Make sure it is exposed to the air for a full 15 minutes, then cover it and bring it back to the lab. Place it UPSIDE DOWN on the middle shelf of the white incubator (to the left side of the wall where the doors are). We will see what you grow out on Thursday. Bring your goggles and gloves from now on.