Microbial Control
Physical Control of Microbes - Heat and Cold
Chemical Antimicrobials - in vitro and in vivo/chemotheraputics /
Part One - Physical Control
Review text and lecture notes about controlling microbial growth using temperature and radiation like UV, understanding both the process and the main target(s) in the cell. For this part of the lab, we will compare spore-forming Bacillus (B) with non-spore-forming Micrococcus (M). In this and other other labs, you will be plating larger volumes using a new tool called a spreader. You will also learn how to alcohol/flame-sterile steel tools like forceps and spreaders.
Procedures - Cold/Refrigeration
Understand alcohol/flaming; do not hold dipped tools in flame longer than it takes to light them!
Label 2 nutrient agar plates, 1 for B and 1 for M - spread 0.2 ml of each culture on each plate
Place both plates at 14°C (i.e. assigned location of refrigerator)
Procedures - Pasteurization
These cannot be done as above because plates would melt if grown at higher temperatures!
Label 2 sterile test tubes and 2 nutrient agar plates, 1 set for B and 1 set for M
Aseptically add 0.5 ml of each culture to each tube and place in 71°C water bath for 15 seconds
After heating, spread 0.2 ml of each culture on each plate; grow in 37°C incubator
Procedures - UV-Irradiation
Label 2 nutrient agar plates, 1 for B and 1 for M - spread 0.2 ml of each culture on each plate Place plates in the UV area when directed - with lid half off and half on (note which side is which)
Your instructor will UV-expose the plate and return it by the end of lab; grow in 37°C incubator
Part Two - Testing Antimicrobial Chemicals
Review text and lecture notes about antiseptics and disinfectants. Today, you will test chemicals used in vitro on Gram (-)Pseudomonas/ (P) and Gram (+) Staphylococcus/Streptococcus (S). Antimicrobials must be placed on sterile filter disks prior to use. The most effective way of doing this is to place 1 DROP of each compound in a sterile petri dish and then gently lay the disk in the substance; avoid overloading the paper because drippy disks will mess up your results. Impregnated disks will be applied to lawns of microbes that you make, allowed to grow, and then measured in terms of “zones of inhibition.”
Procedures
Obtain 2 nutrient agar plates – label carefully.
For each culture, test 4 compounds (3 chemicals, 1 water) - all 4 on 1 plate
Label and divide each plate into 4 sectors; spread 0.2 ml of culture onto each plate
Remove filter disks with alcohol/flame-sterilized tweezers - dip in selected antimicrobials
Load non-drippy disks onto appropriate sectors; grow in 37°C incubator; examine next week
Part Three - Testing Antimicrobial Chemotheraputic Agents
Review text and lecture in terms of chemotheraputic drugs meant to be taken by people to combat disease. These compounds include both antibiotics and synthetic compounds. This exercise will allow you to test specific drugs from lecture - plus new ones -. In contrast with the last exercise, provided disks have been commercially impregnated with drugs.
Procedures
Teams will not be provided with extra disks because they are extremely expensive!
Obtain 4 nutrient agar plates – 2 for each microbe to be tested
Label media-containing base of each plate with your initials and test organism
For each organism, test 6 different drugs - 3 on each plate
Label and divide each plate into 3 sectors; spread 0.2 ml of culture onto correct plates
Place disks onto appropriate sectors, making sure you understand what each contains
If any look loose, tap gently with your alcohol/flame-sterilized tweezers
Load non-drippy disks onto appropriate sectors; grow in 37°C incubator; examine next week
Biology 318 Worksheet Due Next Lab - Turn in Individually
Name:
(1) 2 pts. Temperature Control.
Describe results – if colonies <100, count; >100 = report TMTC (too many to count).
Condition / Data-M / Data-BRefrigerator
Pasteurize
(2) 2 pts. Describe results for organisms in terms of UV exposure testing.
M:
B:
(3) 3 pts. What targets in the cell do heat and UV damage?
HEAT:
UV:
(4) 2 pts. Why you were expecting to see differences between M and B. Did you? Explain.
(5) 6 pts. Antiseptics and Disinfectant Testing.
You will have to use the text and product labels; (mm) - zone radius in mm.
Chemical/ Active Ingredient(s) / Cell Target / S (mm) / M (mm)Water, none - control / None - control
(3) 6 pts. Chemotheraputic Agents. Use your text to complete information about the new drugs tested, as modeled by my answers for those from lecture.
Drug / Source / Cell TargetPenicillin / Penicillium, Fungi / Cell Wall
Sulfa / Synthetic / Folic acid synthesis
Streptomycin / Streptomyces, Bacteria / Protein synthesis
(4) 9 pts. Complete and use this table to determine whether each drug tested performed as it should have (i.e. did the data you observe match the spectrum)
Drug / Spectrum / (mm) / (mm) / Match Interpretation - ExplainPenicillin / Limited/Gram (+)
Sulfa / Broad
Streptomycin / Broad
Biology 318, Lab Three, 1