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BI-304 Environmental Microbiology W15

Lab #2b: Bacteria and Actinomycetes


(Adapted from: Environmental Microbiology: A Laboratory Manual, Second Edition, Ian L. Pepper and
Charles P. Gerba, Elsevier Science, 2004)

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

THEORY AND SIGNIFICANCE:


Soil bacteria are the most abundant organisms found in surface soils. These organisms are
very diverse; all are prokaryotic, but bacteria can be aerobic, anaerobic or facultatively
anaerobic. In addition, there are autotrophic and heterotrophic bacteria. Within this
prokaryotic group are the filamentous microbes known as actinomycetes. Bacteria and
actinomycetes are important in nutrient cycling and degradation of organic contaminants.
In addition, they interact with plants as rhizosphere populations in close proximity to
plant roots. Finally, soil bacteria can be pathogenic to plants (Agrobacterium
tumefaciens) and humans (Clostridium perfringens and Bacillus anthracis).

Although formally classified as bacteria, actinomycetes are often thought of as an


intermediate between bacteria and true fungi. They are characterized by fine hyphae
(usually less than 1.0 m in diameter) that readily break into fragments resembling
bacterial cells. They are Gram-positive. Most of them are non-spore-forming and non-
motile, and often members of the genus Streptomyces. Actinomycetes play an important
role in the Carbon Cycle as they are involved in the degradation of cellulose and chitin
(plant matter) in soil. They are also well-known producers of antibiotics; Dr. Selman
Waksman was awarded the Nobel Prize in 1952 for his discovery of actinomycin. This
was the first of hundreds of antibiotics to be isolated from these soil bacteria. Unlike

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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

Materials (per pair)


10 g rich garden soil
bench-top balance
6 peptone-yeast agar plates
6 actinomyces isolation agar plates with 0.3 mg/mL cyclohexamide (antifungal agent)
1 x 95 mL sterile water blank
4 x 9 mL sterile water blanks
1mL sterile pipettes
Ethanol and hockey sticks for spread plates
Vortexes

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BI-304 Environmental Microbiology W15

Protocol (per pair)


A) Bacteria
1. Prepare a 10-1 dilution of the rich garden soil sample by adding 10 g of soil to 95 mL
of sterile water. Shake well.
2. BEFORE THE SOIL SETTLES TO THE BOTTOM OF THE TUBE further dilute
your sample to a final concentration of 10-5 using a 10-fold serial dilution. Vortex well at
each step, and make sure that the soil has not settled before performing each dilution step.
3. To enumerate the bacteria in the soils, prepare duplicate spread plates (0.1 mL
inoculum) for each of the 10-3, 10-4 and 10-5 dilution tubes from soil sample on peptone-
yeast agar plates.
4. Incubate the plates, inverted, at room temperature for one week.

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

Materials (per pair)


incubated plates from week 1
5 peptone-yeast agar plates

Protocol (per pair)


1. Examine all of the bacteria plates carefully. Note differences in colony shape and size.
2. Count the colonies on any countable (20-200 range) plates and calculate the cfu/g of
your soil samples. Include all types of growth in your count/calculation.
3. Select five well-isolated individual colonies from any of the plates. Try and include an
actinomycetes colony as one of your five.
4. Prepare purity plates of each of these colonies by streaking for isolation on a peptone-
yeast agar plate.
5. Incubate these plates, inverted, at room temperature for one week.

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BI-304 Environmental Microbiology W15

Week 3

Materials (per pair)


incubated actinomycetes plates from week 1
incubated purity plates from week 2
2 x sterile microfuge tubes
sterile water in a capped test tube
2 peptone-yeast agar plates
antibiotic disks
vortex
alcohol and hockey stick spreaders

Protocol (per pair)


A) Actinomycetes plates
1. Examine all of your actinomycetes plates carefully. Note differences in colony size and
shape.
2. Count the total number of actinomycete colonies on plates that are in-range (20-200).
Do not count any simple bacterial colonies.
3. Calculate the cfu/g of your soil sample.

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

Lab 2: Full Report WRITE-UP INSTRUCTIONS


Due in Week 7 (Week of Feb 23rd)

1. Objectives: clearly state the overall and specific objectives of the experiment /4

2. Materials & Methods: /4


Signed pre-lab flow charts for Lab 2a and 2b
Notify any changes to procedure

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

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