INTRODUCTION Waste digester is all natural microencapsulated bacteria and enzyme widely used in aquaculture and claimed to eliminate odor and dissolve organic waste in ponds, lagoons, septic and cesspools.

OBJECTIVES:

1. To identify the different functional groups of bacteria present in waste digester.

2. To enumerate and compute biomass in terms of cells ml^-1 of the different functional group and their total biomass.

METHODOLOGY:

I. Enumeration of Functional Groups of Bacteria in Waste Digester

A. Preparation of Sample

1. Dissolve 25g probiotics in 225 ml of brackish water diluent (15ppt). Mix thoroughly to have a homogeneous mixture. This represents your 1:10 dilution.

2. From 1:10 dilution transfer 10ml to the first tube and mix thoroughly by pipetting in and out thrice. This represents 1:100 dilutions. From this dilution, transfer 10 ml to the 2^nd tube and mix thoroughly to have a 1:1000 dilution. The procedure is repeated for 3^rd and 4^th tube, to yield 1:10,000 and 1:100,000, respectively.

3. Set aside for plating.

B. Total heterotrophic bacteria

1. 0.1 ml from 10³, 10⁴ and 10⁵ dilution is pipetted and introduced into seawater agar plates in 3 replicates. Spread the sample using a flamed L-rod.

2. Incubate for 18-24h at room temperature.

3. Enumerate the different colonies grown in nutrient agar and record morphological characteristics.

4. Compute for viable count in terms of colony-forming units (cfu ml^-1).

Average colonies per plate

CFU ml^-1 =

Volume plated (ml) x DF

A. Total Vibrio count

1. 0.1 ml from 10¹, 10² and 10³ dilutions is pipetted and introduced into TCBS agar in 3 replicates. Spread the sample using a flamed L-rod.

2. Incubate for 18--24h at room temperature.

3. Enumerate the different colonies in TCBS agar and record morphological characteristics.

4. Compute for viable count in terms of colony-forming units (cfu ml^-1).

Average colonies per plate

CFU ml^-1 =

Volume plated (ml) x DF

D. Total Pseudomonas

1. 0.1 ml from 10¹, 10² and 10³ dilutions is pipetted and introduced into Cetrimide agar in 3 replicates. Spread the sample using a flamed L-rod.

2. Incubate for 18-24h at room temperature.

3. Enumerate the different colonies grown in Cetrimide agar and morphological characteristics.

4. Compute for viable count in terms of colony-forming units (cfu ml^-1).

Average colonies per plate

CFU ml^-1 =

Volume plated (ml) x DF

E. Total Mold and yeast count

1. 0.1 ml from 10¹, 10² and 10³ dilutions is pipetted and introduced into Saboraud agar. In 3 replicates. Spread the sample using a flamed L-rod.

2. Incubate for 18-24h at room temperature.

3. Enumerate the different colonies grown in Saboraud agar and morphological characteristics.

4. Compute for viable count in terms of colony-forming units (cfu ml^-1)

Average colonies per plate

CFU ml^-1 =

Volume plated (ml) x DF

F. MPN Nitrate

1. Preparation of sample. The same procedure outlined in I.A is followed but with some modification. I ml from each dilution is serially diluted up to 10^-9.

2. Using a flamed forceps, aseptically insert one fermentation tube in each dilution tube, bottom up to capture the gases being evolved (which may include molecular Nitrogen). Be careful not to create bubbles in tubes.

3. Incubate the tubes at 35^oC for 24-48h.

4. Add 1.0 ml each of nitrite reagent (sulfanilic acid and alpha-napthylamine) directly to the tube. A positive result (red) color within 30 seconds denotes a complete test. If result is negative. Add a pinch of nitrate –free zinc dust to the tube and color development is observed within 30 seconds.

5. Determine MPN by multiplying the minimum positive tube with the dilution factor.

6. Plate 0.1 ml of the least dilution for characterization of the colonies present.

III. Observe growth of colonies from the different media and note their morphology. A colony is composed of descendants of single cell and is nearly visible to the naked eye. Colony may be described as to their size, shape, color, texture, surface, margin, elevation, odor, etc.

A. Examine colonies under 4x magnification of microbiological microscope or 6.46 and 16x of stereoscope. You may use camera lucida for your drawings.

B. Maintenance of Culture

i. Subculture each colony in separate NA slants. Label them with their corresponding number of colonies. Note growth of slant after 18-24 hours incubation.

ii. Reserve slants for the next exercise.

RESULTS AND DISCUSSION

Guide Questions

1) How many types of colonies grow on your plate culture media? Why is it that some media encourage growth of microorganisms and others inhibit growth?

2) What are the functional groups of media? Discuss their functions.

3) Tabulate the morphological characteristics of the colonies in different media and make some drawings

Laboratory Exercise No. 5 Functional Groups of Bacteria

RESULTS:

Table 1. Viable counts of different functional groups of bacteria

Functional Groups

No. of colonies

Cfu ml-1

Dil

Heterotrophic

bacteria

Vibrio

Pseudomonas

Mold

Yeast

Table 2. Morphological Characteristics of bacteria from different functional groups

Functional groups	Viable count	MORPHOLOGICAL CHARACTERISTICS OF MICROBIAL colonies
Heterotroph		Size	Shape	Color	Txt	Elev	Sur	Mar	Odor
Isolate 1
Isolate 2
Isolate 3
Isolate 4
Pseudomonas species
Isolate 1
Isolate 2
Molds and Yeasts
Isolate 1
Isolate 2
Vibrio species
Isolate 1
Isolate 2
Sulfur reducers
Isolate 1
Isolate 2
Nitrate reducers
Isolate 1
Isolate 2
Nitrite reducers
Isolate 1
Isolate 2

Marine biology lectures and activities

References

Friday, June 3, 2011

Laboratory Exercise No 5. Functional Groups of Bacteria

INTRODUCTION Waste digester is all natural microencapsulated bacteria and enzyme widely used in aquaculture and claimed to eliminate odor and dissolve organic waste in ponds, lagoons, septic and cesspools.

OBJECTIVES:

Laboratory Exercise No. 5 Functional Groups of Bacteria