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.
Get a white tray from the back of the room and always put your plates and racks of tubes on the tray so they are not directly on your desk top. When staining a slide, get a grey bucket from the back of the room and place it on the white tray. Then get a 250 ml beaker, place it in the grey bucket. Place your slide on top of the beaker so the beaker catches most of the stain that drips off it. The grey bucket will catch the rest of the stain. The stain waste is then dumped into the white containers on top of each of the two lab rows.
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, etc. Use the regular trash for everything else.
For the glassware flasks and tubes put them on the indicated tray on the left side of the room.
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:
INSTRUCTIONS FOR TODAY
Download all of the instructions for the labs this semester from my website, and save them in a folder. Make sure you bring ALL the semester’s instructions with you each week, because we will use the instructions from previous weeks throughout the semester.
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 NAPetri dishes per lab group. The lid is the larger (wider) side and the bottom is the smaller side. Use the Sharpie or wax pen to write your group leader 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 water to it, and swab a surface to collect bacteria, one swab 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 Yellow Petri Dish rack except the sample from your body will go in the Red Petri Dish rack, which goes in the incubator. The nutrient broths will go into the incubator in one rack. Nexttime, 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 (soil sample after dinner)
- 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 or plastic rod bent at 90°.
4)Pour Plate
- 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
Moisten a cotton swab with water and obtain one different fomite sample (inanimate object) for each of the two nutrient agarstreak plates. For example, swab your phone and inoculate one plate and then another person uses 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 (unless you are swabbing mouth or throat, the swab can be dry) 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 counterclockwise 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 top of the second quadrant, then (without lifting it) zig-zag throughout the second quadrant, keeping thezig-zags close together. Throw the swab out. Turn the plate counterclockwise so quadrant 3 is at top. Get a new dry swab and drag it in one line from themiddle 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, turn the plate counterclockwise, 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 see 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 (next lab period) 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 body swabs at 37° C, which is body temperature. The others will be room temperature.
HANDWASHING WITH SALIVARIUS AGAR
Streptococcus salivarius is a species of Gram-positive cocci bacteria which colonize the mouth and upper respiratory tract of humans a few hours after birth, making further exposure to the bacteria harmless. These bacteria are opportunistic pathogens, causing dental caries and endocarditis in the immunocompromised.Mitis salviarius agar is used to differentiate among species ofStreptococcusthat are flora in the mouth. The sugars in this medium are sucrose and glucose. The medium also contains the dyes trypan blue and crystal violet, which inhibit other Gram positive and Gram negative bacteria. S. salivarius is able to use the sucrose in the media to produce a capsule around itself.Strep salivariususes the sugars to produce a gummy-like capsule, producing sticky, mucoid, gum-drop colonies.Strep mitiscolonies will be small, flat, light blue.Strep mutanswill be undule-shaped colonies, with a granular frosted-glass appearance (making dextran from sugar). Enterococcuswill produce dark, blue-black colonies.
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 all the way up with soil.
3)Record the place of collection, temperature, and any of the below data you can collect
LOCATION
Location
Latitude
Longitude
Date and Time Collected*
Date
Time
Sample Site Descriptors*
SOIL SAMPLE
Air Temperature (°C)
Humidity (%)
Depth (In.)
Type of Soil
Soil Temperature (°C)
pH of Soil
Water Content (%)
4)Pour the whole 15 ml tube of soil into a 250 ml Erlenmeyer flask and fill it to the 100 ml mark with de-ionized water in the large plastic jar by the sink. That gives a 15% soil to water ratio. Stir it up to make it muddy.
5)EACH STUDENT will obtain one of each of the following two plates PER SOIL SAMPLE: LBA (Luria-Bertaniagar), and TSA (tryptic soy agar)
6)Label the BOTTOM side of the Petri dishes, around the EDGE, with your name, “soil”, date, and instructor name.
7)Use a pipette to drop 0.5 ml of the solution (half a stem full of the pipette) onto a Petri dishby opening the lid of the Petri dish at a 45° angle, keeping the lid over the agar.Squirt the solution onto the top of the agar.
8)Use a sterile spreader, but you can use the same spreader for each of the three plates from the same soil sample. Spread across the whole plate surface while rotating the plate to be sure the whole surface is covered.
9)Make sure your plates all face upwards when you wrap your three plates togetherwith parafilm(don’t wrap each plate individually) and add a piece of tape on top with your name on the tape. Then turn them upside down so the agar is on top. Leave theseat room temperature (yellow Petri dish holder). Do NOT put these in the incubator. The Tech will put them in the refrigerator on Monday so they do not overgrow.
10)Clean up: throw out muddy water into the regular trash can. Put the muddy flask on the tray to the left of the sharps container. Put your blue top plastic tubes that your soil was in. Rinse the plastic spreaderand put in on the tray where the flasks are.
HOW TO REMOVE YOU GLOVES
HAND WASHING WITH GLOGERM™
- Shake the bottle well.
- Have your lab partner apply 5-6 drops of the gel on the palms of one of your hands.
- Rub your hands together so the lotion covers all surfaces, including backs, between the fingers, and up to the wrists. Scratch the palms with your fingernails.
- Have your lab partner shine your hands with an UV-lamp to see the extent of coverage. Do NOT shine U.V. light into anyone’s eyes!
- Ask your partner to turn on warm water at the sink for you. Wash your hands with warm water and soap the way you usually do.
- Have your lab partner shine your hands with an UV-lamp to see the extent of coverage. Ask your partner to record your observation in the table.
- Repeat your hand washing in a thorough way, scrubbing your hands for at least 20 seconds.
- Have your lab partner shine your hands with an UV-lamp to see the extent of coverage.
- Repeat the procedure with your lab partner but with the roles reversed.
HOW TO WASH YOUR HANDS PROPERLY
It takes at least fifteen seconds to wash your hands properly – this is about how long it takes to sing ‘Happy Birthday to You’ twice through!
Before you wash your hands, take a paper towel and put it under your arm.
Wet hands with water and leave the water running
Apply enough soap to cover all hand surfaces
Rub hands palm to palm, rub between fingers, and backs of hands, fingers, and wrists.
Rinse hands with water and do not turn the water off while drying your hands.
Dry thoroughly with the towel from under your arm.
Use the same towel to turn off the water faucet.Use the towel to open the bathroom door, and then discard the towel in trash and exit without touching anything else.