Introduction:
Microbes exist mixed with other microbe populations. In order to study and identify organisms, it is necessary to isolate a pure culture in which a
ll organisms are descended from the same organisms. Organisms must be grown in a sterile environment free of all other organisms to be studied, and grown on a medium containing nutrients. A medium is generally sterilized by heating it to a temperature which destroys all organisms. Methods for handling organisms to avoid contamination is generally referred to as the aseptic technique.
Cultures & Media:
Broth tubes contain a liquid medium. A common broth is trypticase soy containing nutrients for microbial growth. Growth can be seen as a pellicle (mass of floating organisms), turbidity of the broth, or a sediment of organisms on the bottom.
Slant tubes contain medium solidified with again at an angle for a flat surface to inoculate. Stab tubes are deep tubes of a solid medium inoculated by stabbing. Agar plates contain a thin layer of solid
medium, and may be used to culture, separate, and count microbes.
Incubation:
Obligate aerobes: grow in the presence of oxygen only, aerobic respiration
Obligate anaerobes: grow only without oxygen, oxygen inhibits growth or kills them, typically use anaerobic respiration or fermentation
Aerotolerant anaerobes: do not use oxygen but tolerate it fairly well, energy from fermentation
Facultative anaerobes: grow with or without oxygen, but generally better with oxygen, energy from aerobic respiration, anaerobic respiration, and fermentation, most bacteria
Psychrophiles: optimum growth at -5 to -15 degrees Celsius, live in arctic & Antarctic regions
Mesophiles: optimum growth at 25-45 degrees Celsius, live in soil, in & on the body
Thermophiles: optimum growth at 45-70 degrees Celsius, live in hotsprings, compost
Hyperthermophiles: optimum growth at 70-110 degrees Celsius, Archaea bacteria, live near thermal vents in ocean
Colony appearance:
A visible mass of organisms growing on a medium is a colony. Appearance may vary in color, elevation, surface, & form. Water soluble pigm
ents diffuse out of the microbe into the surrounding medium, while water insoluble pigments do not diffuse into the agar. Organisms that produce pigment during growth are chromogenic.
Staphylococcus aureus: gold, water insoluble
Micrococcus luteus: yellow, water insoluble
Micrococcus roseus: pink, water insoluble
Mycobacterium phlei: orange, water insoluble
Serratia marcen
scents: orange / red, water insoluble
Pseudomonas aeruginosa: green / blue, water soluble
Procedures:
Aseptically inoculate 1 trypticase soy broth tube with the sample culture from a culture tube.
1. Sterilize inoculating loop by dipping it in the sand jar, and then passing through the flame of a gas burner until the wire becomes red from heat. Allow it to cool to avoid killing your inoculum. DO NOT LAY THE LOOP DOWN ONCE IT IS STERILIZED OR IT MAY BECOME CONTAMINATED.
. Remove the inoculum by holding the culture tube in 1 hand and the loop in the other. Remove the cap off the culture loop with your pinky finger to avoid laying it down. Briefly flame the opening of th
e culture tube. Insert the loop & remove a sample of inoculum. Flame the culture tube opening again and replace the cap.
3. Remove the cap from the sterile broth medium & flame the opening of the culture tube. Insert the loop with the inoculum into the broth & briefly move it around. Flame the opening of the tube prior to replacing the cap.
4. Sterilize the loop again by dipping it in the sand jar and passing it through the flame.
Inoculate
an agar plate from a sample of a culture broth of the sample culture
1. Sterilize the loop.
2. Use aseptic technique to open the broth tube, insert the loop to obtain a sample of the culture and replace the cap using aseptic technique. Lift the edge of the petri plate enough to insert the loop and streak the sample. Keep the loop near parallel to the
surface to avoid digging into the agar. Streak for isolation.
a. “Streaking a plate” starts in 12:00 position and streak side to side as your pull the loop toward yourself. You will continue to streak about half way down the plate.
b. Sterilize the loop using aseptic technique. Pass ONE time through the previously streaked half of the plate, and then streak the next quarter section of the plate pulling the loop toward yourself.
c. Sterilize the loop again using septic technique. Pass ONE time through the previously streaked quarter of the plate, and then streak the last quarter section of the plate pulling the loop toward yourself.
d. Sterilize the loop prior to setting it down to avoid contaminating your work area.
TO STREAK A NUTRIENT AGAR
PLATE FOR ISOLATION OF MICROBES:
Inoculate 1 trypticase soy agar stab tube with a sample from the culture tube.
a. Use the same aseptic technique with dipping into the sand jar and flame to sterilize your needle.
b. Use aseptic technique to open the culture broth & dip the sterile needle into the culture. Replace the cap using aseptic technique.
c. Open the slant tube using aseptic technique by flaming the opening of the tube. Insert the inolculated needle into the tube and stab it deep into the agar, and then drag the needle across the top. Flame the opening of the stab tube prior to replacing the cap.
d. Sterilize the needle before setting it down to avoid contamination of the workspace.
Other activities for inoculation:
Inoculate ½ of 1 trypticase soy again plate with organisms from your skin by rubbing your finger over ½ of the plate. Streak the remainder of the plate for isolation.
Inculate ½ of 1 trypticase soy agar plate with organisms from a hair from your head by dragging the hair over the plate. Streak the remainder of the plate for isolation.
Incubate all of the tubes and plates at 37 degrees Celsius. Place the tubes in the beaker or test tube rack to keep them upright. Incubate plates upside down to prevent water condensation. Make sure a
ll tubes are marked with a wax pencil before incubating. Always label the bottom of plates in case they become separated from their lids.
Take home: 3 volunteers will take home 1 plate each and place it in the bathroom. While depositing feces in the toilet, flushing the toilet, and completing your activities in the bathroom, place the lid on the dish and allow it grow at room temperature until the next lab session. The 3 plates will be viewed by the entire class and observe for growth.
Observations following incubation:
Broth Culture: Is the the culture turbid? Does it form a pellicle? Is there sediment on the bottom? What is the color?
http://microbiologya.blogspot.com/
Streak plates: How many different colony types are on each plate? What is the elevation of the colony? Does it have a smooth or rough margin or edge? Is the surface smooth, rough, fuzzy, slimy, mucoid etc? Is there any pigmentation to the colony? Is there pigment solubility - does it appear to release pigment into the surrounding media?
http://inst.bact.wisc.edu/inst/index.php?module=Book&func=displayarticle&art_id=119
https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEj6UqjCbTmlCnxxGiFulNayFG2z8rsUHS8gL6peLyjUW7OMZFOSWtgb4xsy_pQoFDQkQR3d0RhDAv2WwQTSK8mlCUEbd2Df74rdqGoYk4Hzj7f3_4bRiWFWiG3mqJPVCpZ5_28cVhRiZCU0/s320/pyocyanin.jpg
Stab tubes: Describe the growth. Does it appear to have more growth on the surface or the bottom? Does the growth stay in the line of the stab, or does it appear to diffuse out from inoculation point?
http://www.uwyo.edu/molb2210_lab/info/biochemical_tests.htm
