Shalasai Huangprasert
Collecting Samples
Calculating Dry Weight
Culturing Microorganisms
Preparing Slides of Microorganisms
Collecting Samples
Microorganisms are not distributed uniformly throughout compost; they
commonly occur in clumps or colonies ranging from few to thousands of individual
cells. The populations vary greatly depending upon the amount of undecomposed
organic matter and the micro-environment in a specific location. How wet
the sample is, and whether it contains anaerobic or aerobic regions, also
will affect the types of microbial life that are found. Multiple samples
therefore should be taken to determine microorganism numbers or activity
in compost.
Calculating Dry Weight
Water content of different composts may vary greatly. When comparing
how much microbial activity there is in a gram of compost, you must allow
for the difference in water content so that you can accurately compare
what is happening in equal amounts of two different composts while discounting
the weight of the water.
To determine the ratio of wet to dry weight of a compost, a sample of
wet compost is weighed and then dried for 24 hours in a 105-110C oven.
It is then reweighed, and the ratio between wet and dry weight is calculated.
When using the actual (wet) compost in a study, the moisture ratio is
used to calculate how much compost to use. For example:
Amount of vegetable waste compost needed = 5 g
Predetermined wet weight of a sample = 4.3 g
Measured dry weight of the same sample = 2.8 g
Ratio of wet/dry = 1.54
Actual amount of compost needed for the experiment would be:
5 g x 1.54 or 7.7 g (wet weight)
Culturing Microorganisms
The procedures to use in culturing micooganisms depend on which types
of organisms you wish to study.
Bacteria
Actinomycetes
Fungi
Culturing Bacteria
To culture bacteria, the following media should be used to prepare agar plates:
Growth Media: 1/10-strength Trypticase Soy Agar (TSA) Media:
Ingredients:
2 g Trypticase Soy Agar
7.5 g Bacto Agar
500 ml distilled water
Mix the ingredients, autoclave for 20 minutes, and pour into sterile petri dishes.
Plate out the bacteria using a 10-7 dilution starting with 5 g dry weight of the compost in 45 ml of an autoclaved .06M NaHPO4/NaH2PO4 buffer of 7.6 pH. (approximately 4:1 dibasic:monobasic). Put this first dilution in a blender for 40 sec. at high speed.
Perform serial dilutions to 10-7 and add 0.1 ml of the dilution per plate. Incubate at 28C for 4 days.
Count the colonies as colonies per unit after 4 days. Prepare slides of specific colonies the same day.
Growth Media: 1/50-strength TSAPoly B
Ingredients:
0.4 g Typticase Soy Agar
10.0 g Bacto Agar
500 ml distilled water
10 mg Polymixin B in 10 ml 70% Ethanol
Mix the first 3 ingredients, autoclave for 20 minutes, and cool to room
temperature. Add the antibiotic and pour into sterile petri dishes.
Plate out the actinomycetes using a 10-7 dilution starting with 5 g
dry weight of compost in 45 ml of the autoclaved buffer. Put this first
dilution in a blender at high speed for 40 sec.
Perform serial dilutions to 10-7 and add 0.l ml of the final dilution
to each plate.
Incubate the plates at 28C for 14 days.
Take counts and samples of actinomycetes colonies after 14 days. Many
of the colonies will look powdery white. However, some may take on a rough
appearance and produce a variety of pigments.
Note: If you are comparing mesophilic compost to thermophilic compost, you should prepare double the usual number of plates so that you can incubate plates at both 28C and 50C.
Culturing Fungi
Growth Media: 1/3-strength PDARP
Ingredients:
6.5 g Potato Dextrose Agar
5.0 g Bacto Agar
500 ml distilled water
15 mg Rifampicin in 10 ml Methanol
15 mg Penicillin G in 10 ml 70% Ethanol
Mix the first 3 ingredients, autoclave for 20 min. and cool to room
temperature. Add the antibiotics and pour into sterile petri dishes.
Plate out the fungi using a 10-4 dilution starting with 5 g dry weight
of compost in 45 ml of the autoclaved phosphate buffer. Put this first
dilution in a blender at high speed for 40 sec.
Perform serials dilutions to 10-4 and add 0.l ml of the final dilution
to each plate.
Incubate the plates at 28C for 3 days.
Take counts and samples of fungal colonies at 3 days.
Preparing Slides of Microorganisms
Bacteria
Use sterile techniques to prepare your slide! Use an inoculating needle
to add a drop of saline to a clean slide. Take a sample of a single bacterial
colony and mix it into the saline. Let air dry until a white film appears.
Heat fix by passing the slide through a flame a few times.
Actinomycetes
Follow the above procedure but try to get a portion of the colony on
the slide intact. You can try lifting it with a sterile scalpel. It will
be too crowded to observe on most of the slide, but at the edges of the
colony you will be able to see the pattern that the filaments form.
Fungi
Lift a portion of the colony intact onto a clean slide (it will still
be attached to the agar), add a cover slip and observe without staining.
Look at the edges of the colony where the sample will be thinner and there
will be enough light to observe.
Staining Slides
Gram staining can be used to make slides of bacteria and actinomycetes:
Preparation of Gram Stains
Crystal Violet:
Dissolve 2 g of crystal violet in 20 ml of 95% ethanol. Add this solution
to 80 ml of a 1% Ammonium Oxalate solution. Let stand for 24 hours and
filter.
Gram Iodine:
Add 1 g Iodine and 3 g Potassium Iodide to 300 ml distilled water. Store
in an amber bottle.
Decolorizer: 95% Ethyl Alcohol
Safranin:
Add 2.5 g safranin to 10 ml 95% ethanol. Add this solution to 100 ml
distilled water
Procedure for Gram Staining
1. Flood slide with crystal violet - 20 sec.
2. Wash with distilled water - 2 sec.
3. Flood slide with Gram iodine - 1 min.
4. Decolorize by tilting slide and drop by drop rinsing with 95% ethanol until
ethanol runs clear - about 10 to 20 sec.
5. Wash with distilled water - 2 sec.
6. Flood with safranin - 20 sec.
7. Wash with distilled water - 2 sec.
8. Blot dry.