LAB SAFETY: Open-toed shoes and shorts above the knee are not allowed since a spill can cause skin irritation. Put your backpack, purse, etc in the racks at the back of the room. Before leaving, make sure your area has been disinfected with desktop cleaner, left clean, push your chairs in, and that the microscope has been put away properly. If it is found not properly put away, you will receive a note indicating what was wrong with it.
The lab counters must all be wiped down with disinfectant when you come in and when you leave. You may throw the paper towels into the regular trash afterwards.
Dispose of cover-slips and glass slides in the red sharps container. Most fires in this lab are started from alcohol becoming ignited. Just get away from it, cover it, and let it burn out. Remember, a lab coat can be taken off and used to put out a fire if someone’s hair catches fire, etc.
There is no food, drink, gum or water bottles allowed in a micro lab.
The biohazard bag is for any and all hazardous materials, including a toothpick you put in your mouth, gloves, disposable Petri dishes, etc. Use the regular trash for everything else.
For the glassware flasks and tubes, make sure all labels are removed, and all grease marks are erased first.
If there is a bacterial spill, cover it with paper towels, saturate them with disinfectant (Clorox) for ten minutes, then put those paper towels in the biohazard bucket.
First aid kit is in the cabinet near Lab Tech door.
Recent case of Salmonella infection that came from a teaching lab:
http://www.cdc.gov/salmonella/typhimurium-laboratory/011712/index.html
INSTRUCTIONS FOR TODAY
Divide into groups of 3-4. Each GROUP will get one nutrient broth tube, three nutrient agar plates (Petri dishes), and 4 packs of sterile swabs (2 per pack). One person will be the group leader who will put their name on the plates and tube.
HOW TO INOCULATE THE BROTH
Label the tube with your group leader’s name and source of the swab. To inoculate the tube of nutrient broth, moisten a cotton swab with water, swab the inanimate surface (fomite) you want, then stick the whole swab in the tube and swish it around. Press the swab against the inside of the glass to squeeze off the excess liquid, and discard the swab in the regular trash. Twist the cap closed, then open it by ½ turn so air can get in. Place it in the rack with the tubes from the rest of the class, and the whole rack of tubes will go in the incubator.
Take three TSA Petri dishes per lab group. The lid is the larger (wider) side and the bottom is the smaller side. Use the Sharpie pen to write your group name on the bottom of the Petri dish, around the edge in a ring.
On one plate, turn it upside down and draw a giant plus sign on the bottom so the plate is divided into four quadrants. You will get a sterile cotton swab, add a drop of sterile water to it, and swab a surface to collect bacteria for each of the three plates and the tube. The plate with the quadrant should be inoculated with a sample from your body (either skin, throat, hair, etc). The other plates should be a sample from the environment (light switch, computer keyboard, bathroom, water fountain, door knob, etc). Make sure you write your source on each plate and tube. Instructions on how to inoculate the plates are on the next page.
The plates will stay out at room temperature in the Petri Dish rack except the sample from your body will go in the incubator. Next week, we will examine the colonies and practice using the proper terminology to describe the morphology (appearance) in your tube and plates.
PLATE INOCULATION TECHNIQUES
To inoculate the plate, there are four different techniques, depending on what tests you will perform on the subculture. Today, we will do 2 “streak for confluence” plates (on the fomite swabs) and one “streak for isolation” plate on your body swab.
1) Streak for confluence Plate (use this technique today on the 2 fomite swabs)
2) Streak for Isolation Plate (use this technique today on your body swab on the plate with quadrants).
3) Spread Plate
a. A small amount (several drops) of a previously diluted sample is spread over the surface of a solid medium (Petri dish) using a spreading rod, which is a sterile glass rod bent at 90°.
4) Pour Plate
a. A small amount of diluted sample is mixed with melted agar and poured into empty, sterile Petri dishes. A serial dilution of the bacterial sample is performed first, then a small amount of each dilution is pipetted into the empty Petri dishes, then the melted agar is added. This allows bacteria to be inoculate throughout the media.
The spread plate and pour plate techniques are quantitative methods used to determine the number of bacteria in a sample. We will use those techniques later in the course.
HOW TO PERFORM A STREAK PLATE FOR CONFLUENCE INOCULATION
Use your swab to obtain one different fomite sample (inanimate object) for each of the two nutrient agar streak plates. For example, swab your phone and inoculate one plate and then use a new swab on a doorknob and inoculate the other plate. The inoculated swab is used to streak the sample many times in a zig-zag over the surface of the agar in the Petri dish. It is streaked from top to bottom. The streaks should be fairly close together.
· Never leave a culture dish open, even for a short time when viewing colonies of organisms. During your procedure, keep the lid close to the dish, open it only as far and as long as is necessary to accomplish the procedure, and keep the lid between your face (and your germs!) and the agar surface. Open it sideways, not so the opening is in front of you where you can breathe on it.
· Never place a Petri dish lid on another surface. You must hold the lid in your hand during the transfer. Make sure the open side of the lid is facing downward and covers the agar, so no contaminants can float down into it.
HOW TO PERFORM A STREAK FOR ISOLATION INOCULATION
(Do this on your plate with the quadrant drawn in)
Purpose of this technique: When a doctor takes a swab from an infected wound and sends it to the lab, he needs to know what organisms are in the wound so he can select the proper antibiotic. There usually will be more than one organism present, so the lab will need to take the mixture of bacteria and separate them out into pure cultures before they can identify each organism.
The body swab will be used for the streak for isolation plate. Use the Petri dish with the four quadrants drawn on the bottom (label each quadrant 1, 2, 3, 4 clockwise; put the numbers in the bottom corner of each quadrant so it does not obstruct your view of the organisms next week). Obtain your inoculum on the moistened cotton swab and zig-zag it over the upper left quadrant (1) of the plate. Make sure your zig-zag strokes are close together to get the most bacteria onto the first quadrant. Throw the swab out. Turn the plate ¼ turn so the second quadrant is at the top. Get a new swab (no need to moisten it; keep it dry) and drag it once across the middle of the first quadrant and into the second quadrant, then (without lifting it) zig-zag throughout the second quadrant, keeping the zig-zags close together. Throw the swab out. Get a new dry swab and drag it in one line from the middle of the second quadrant and drag it into the third quadrant, and zig-zag that area. Throw that swab out. Get a new dry swab and drag the last line from the middle of the third quadrant into the fourth quadrant and zig-zag.
When you use this technique, you are dragging fewer and fewer organisms into each quadrant, so that the last quadrant will have individual colonies that do not overlap each other. If you were successful, in the next lab period you will separate colonies in the last quadrant. If you were not successful, different looking colonies will still be overlapped in the last quadrant. What might have gone wrong? Perhaps you had too many organisms on the first quadrant, or maybe you dragged too long of a line from one quadrant to the next, or perhaps your zig-zags were too narrow and did not cover enough surface area.
The next step (after a few days) would be to select the very best looking individual colony which is separated from the other colonies, touch it with a sterile needle, and zig-zag it across the surface of a slant. Do the same for each of the other colonies that look different from each other, so you end up with one slant for each organism. You would then let those grow and run some tests on them to determine what organisms are present.
INCUBATION
Always incubate plates upside down, so the agar is on the top. Why do we do this? It prevents condensation from developing on the inside lid, and then dropping onto your agar, mixing the organisms. It also enables you to read the important writing on the bottom of the plate. We will incubate at 37° C, which is body temperature.
SOIL PROJECT
1) Don’t use really fertile soil…dry is better, but not in an area where no plants are growing. It is best to get soil close to roots of nearby plants, and dig down about 2 inches first.
2) Fill a 15 ml test tube with about 2 cm of soil.
3) Record the place of collection, temperature, and any other data you can collect (vegetation around etc.).
4) Add enough DI water to fill the tube, close the lid and shake.
5) Place the tube on a vortex machine for 30 seconds to shake more vigorously.
6) Label the BOTTOM side of the Petri dish, around the EDGE, with your name, “soil”, date, and instructor name.
7) Use a disposable plastic pipette to dispense 0.25 ml of water from the tube into the Petri dish. This is the equivalent to just filling ¼ of the STEM of the pipette. Open the lid of the Petri dish at a 45° angle, keeping the lid over the agar. Use a sterile spreader tool to spread the liquid over the entire plate. Incubate upside down at room temperature. The Tech will put them in the refrigerator on Monday so they do not overgrow.
LAB LECTURE MATERIAL
GROWTH MEDIUMS
Nutrients are added to agar (a seaweed product) and heated to a liquid, and sterilized. The sterile liquid agar can be poured into a Petri dish and cooled. The Petri dish is then referred to as a “plate.” Sometimes liquid agar is placed in a tube which is tilted 45° while cooling. This is called a slant. Sometimes the nutrient medium is a broth which does not solidify. Tubes with this medium are called nutrient broth tubes. All of these types of mediums are made for the purpose of being inoculated. Intentionally introducing microbes onto nutrient agar and into nutrient broth is known as INOCULATION, which means having a tiny amount of a bacterial culture streaked across the surface. After inoculation, the plate or tube is usually incubated for at least 24 hours to encourage growth of the sample. After 2-3 days, there will be too much growth, so we take it out of the incubator and put it in the refrigerator to slow the growth.
TYPES OF MEDIA
Culture Media Classification
1. By Consistency (broth, slant, or plate)
2. By Contents
- All-purpose (Nutrient agar = NA)
- Isolation
- Selective (selects for only certain types of organisms)
- Enriched (has extra nutrients (blood) for hard to grow species)
- Differential (allows to tell what type of organism is there; changes color)
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 (hard to grow) organisms, so NA is a good all-purpose media. It will support the growth of a wide variety of organisms. It is also inexpensive.
ISOLATION MEDIA
There are three types of media used to isolate particular organisms
1. Selective media
2. Enrichment media
3. Differential media
Selective media
This type of media selects for a particular type of organism to grow. Selective media contain chemicals that prevent the growth of unwanted bacteria without inhibiting the growth of the desired organism. An example is Sabouraud’s Dextrose Agar (SDA), which has a high sugar content and an acidic pH. SDA is called an ISOLATION MEDIA since it 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.