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OBJECTIVE: To isolate, observe and quantify soil bacteria and also to examine
antibiotic resistance of selected isolates.
OVERVIEW:
- serially dilute soil and prepare spread plates on media specific for i) bacteria and ii)
actinomycetes
- count populations of each and obtain pure cultures via streak plates
- analyze pure cultures microscopically (Gram staining) and for antibiotic resistance
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BI-304 Environmental Microbiology W15
simple bacteria, actinomycetes are relatively drought resistant and have an ecological
advantage over bacteria in desert soils, which tend to by dry and alkaline.
Since soils generally contain millions of bacteria and fungi per gram, normally a serial
dilution series of the soil is made after suspending a given amount of soil in buffer or
deionized water. Aliquots of the appropriate dilutions are plated on appropriate media
and, after incubation, the number of colonies on each plate are used to calculate the
colony forming units per gram of soil (cfu/g). This is analogous to the procedure
performed with food samples in Food Microbiology. Simple bacteria and actinomycetes
are easily visually distinguished on agar; bacteria produce slimy colonies while
actinomycetes have a filamentous growth habit. Bacteria will smear under pressure while
actinomycetes will break.
In order to separate the bacteria from the actinomycetes, in this experiment we will use
two different types of media. We will count each population using the serial-dilution and
spread-plate method. From these plates we will generate streak plates with pure cultures.
Finally, we will then characterize the pure cultures via Gram staining and determination
of antibiotic resistance.
Week 1
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BI-304 Environmental Microbiology W15
B) Actinomycetes
1. Using the dilution tubes prepared above, prepare duplicate spread plates (0.1 mL
inoculum) for each of the 10-2, 10-3 and 10-4 tubes on actinomyces isolation agar plates.
Week 2
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BI-304 Environmental Microbiology W15
Week 3
B) Purity Plates
1. Examine your purity plates for uniformity of colony shape and size. Note the presence
of contaminants, if any.
2. Prepare smears from each plate, Gram-stain and examine under the microscope.
3. Note your observations
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BI-304 Environmental Microbiology W15
C) Antibiotic Resistance
1. Transfer 0.3 mL of sterile water in to each of two sterile microfuge tubes. Label one
G+ and the other G-.
2. Select one Gram-positive and one Gram-negative bacterial colony from your purity
plates. Add a small amount of an isolated colony to the appropriate microfuge tube.
Vortex to disperse the cells in the water.
3. Prepare a spread plate from each type of bacteria using a 0.1 mL inoculum.
4. To each plate add three antibiotic disks (each containing a different antibiotic) using
flame-sterilized forceps.
5. Incubate the plates, inverted, at room temperature for one week.
Week 4
Materials
incubated antibiotic plates from week 3
Protocol
1. Examine plates and look for zones of clearing around the disks. Measure and record
the diameter of the zone for each disk. If the zones overlap, use your best estimate.
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BI-304 Environmental Microbiology W15
1. Objectives: clearly state the overall and specific objectives of the experiment /4
3. Results: Present all results in properly labeled figures or tables (table/ figure # with
descriptive titles; describe the results in 2-3 sentences below each table/figure /24
plate counts, colony morphology and calculated cfu/g for soil (4 marks)
sample calculation for cfu/g soil (1 marks)
purity plates (5 marks) marked in the lab on the quality of plates
Gram staining results (5 marks) marked in the lab on the quality of Gram stains
antibiotic resistance (3 marks)
Diagram and interpretation from Contact slide assay (6 marks)
4. Q & A
In what respects are actinomycetes closely related to bacteria. What are the
similarities with fungi? /3
Discuss the mode-of-action and effectiveness of each antibiotic against Gram+ and
Gram- bacteria /5