MICROBIAL DIVERSITY

I. OBJECTIVES & LEARNING OUTCOME

Practice on manipulations for microbial culture in order to:

- Isolate and obtain pure microorganisms by using streak plate technique.

- Isolate and quantify microorganisms by using spread plate technique.
- Identify the microorganism groups (bacteria, actinomycetes, fungi, yeast, microalgae) by colony
characteristics and cell shapes.

II. BACKGROUND

1. MICROBIAL MORPHOLOGY

Colony characteristics

A “colony” is a group of bacteria, fungi, or other microorganisms grown on a solid agar medium.
The cells plated on this medium grow to form a mass. Colony morphology is used to pick out a pure
colony - that is a colony grown from a single parent cell. Morphology encompasses physical
characteristics like form, margin shape, elevation, surface, texture, opacity, and color (Figure 7.1).

Observing the colony characteristics can be done with the naked eye, or it can be imaged with a
camera and image analysis software for automation. Colony morphology is a method that scientists
use to describe the characteristics of an individual colony of microorganisms growing on agar in a
Petri dish. It can be used to help to identify them.

Figure 7.1 Physical characteristics of microbial colonies

 Bacteria

Bacterial colonies are a mass of bacterial cells on a dense medium, isolated from a single
bacterium. All bacteria are genetically similar within the colony and may be considered a clone. Many
colonies of bacteria are spherical or irregular in form. Some others are filamentous or rhizoid
actinomycetes. Many colonies of bacteria are microscopic and are less than 1 mm in diameter. They
are therefore known as punctiform (pin-point). They likewise have a given margin. In order to observe
the tip, the microscope should be used. For the species, the color of the colony varies: white, buff,
violet, purple, etc.

Shapes of bacterial cells:

 coccus (circle or spherical)

 bacillus (rod-like)
 coccobacillus (between a sphere and a rod)
 spiral (corkscrew-like)
 filamentous (elongated)

Figure 7.2 Basic shapes of bacterial cell

Fungi
The fungi form large multicellular aggregates of long branching filaments, called hyphae. There
are vegetative hyphae and reproductive hyphae. Spores are borne on the reproductive hyphae. (Fungal
spores should not be confused with bacterial spores that are resistant bodies formed for bacterial
survival rather than reproductive purposes.) Spore size, shape and structure are used in the
classification and identification of fungi. The tube-like hyphae are responsible for the fluffy
appearance of the macroscopic mold colony. The hyphae and other structures combine to form an
elaborate network called a mycelium
Fungi is a community of eukaryotic species such as yeast, filamentous fungi, and mushrooms.
Under damp and warm conditions, fungi grow well. Based on their morphological and molecular
attributes, they may be categorized. Fungi grow as colonies when grown on stable media. Among
various types of fungi, fungal colony morphologies are different. From fungal colonies, characteristics
such as pigmentation and texture may be observed.
Colonies of fungi are distinct from bacterial colonies. Fungi emerge as textured colonies that are
powdery or fuzzy. Fungi hyphae run across the solid media, producing colonies of rhizoids or
filaments. As tiny oily dots, fungal colonies may not surface. The colors of mycelium and spores also
vary greatly among the fungal species.
On solid nutrient agar, bacterial and fungal colonies can be grown. They display characteristics
to the organism that develops the colony. The color of the colony depends on the kind of
microorganism that the colony produces. It is also possible to use both for the detection of
microorganisms.

Figure 7.3 Fungal colonies (A), fungal hypha (B, C)

Actinomycetes

Actinomycetes are prokaryotic organisms that are classified as bacteria, but are unique enough to
be discussed as an individual group. Actinomycete numbers are generally one to two orders of
magnitude smaller than the total bacterial population. They are an important component of the
bacterial community, especially under conditions of high pH, high temperature or water stress.
Morphologically, actinomycetes resemble fungi because of their elongated cells that branch into
filaments or hyphae. These hyphae can be distinguished from fungal hyphae on the basis of size with
actinomycete hyphae much smaller than fungal hyphae.
Figure 7.4 Actinomycetes colonies (A), actinomycetes hypha (B, C)
Yeast
These are large (5 to 8 µm), single-celled organisms that rarely form filaments. Most yeasts
reproduce by the asexual process of budding. Yeast colonies are usually characterized by a smooth
surface similar to that of many bacteria.

Figure 7.5 Yeast colonies (A), yeast cell (B, C)

Microalgae
Microalgae are microscopic algae invisible to the naked eye. They are phytoplankton typically
found in freshwater and marine systems, living in both the water column and sediment. They
are unicellular species which exist individually, or in chains or groups. Depending on the species,
their sizes can range from a few micrometers (μm) to a few hundred micrometers. Unlike higher
plants, microalgae do not have roots, stems, or leaves. They are specially adapted to an environment
dominated by viscous forces.
Microalgae, capable of performing photosynthesis, are important for life on earth; they produce
approximately half of the atmospheric oxygen and use simultaneously the greenhouse gas carbon
dioxide to grow photoautotrophically. They have chlorophyll and can manufacture their own food
through the process of photosynthesis.
Figure 7.6 Microalgal colonies (A), microalgal cells (B)
III. EXPERIMENTS

Materials required:

 A source of microorganisms (stock culture, previously streaked agar plate or any other
inoculum)
 Inoculation loop
 Spreader
 A striker/lighter
 Alcohol burner
 Agar plate
 Paper towels
 Microscope
 Microscope slides

1. STREAK PLATE TECHNIQUE

Principle of Streaking

The sample/inoculum is diluted by streaking it across the surface of the agar plate. While
streaking in successive areas of the plate, the inoculum is diluted to the point where there is only one
microbial cell deposited every few millimeters on the surface of the agar plate. When these lone
microbial cells divide and give rise to thousands and thousands of new microbial cells, an isolated
colony is formed. Pure cultures can be obtained by picking well-isolated colonies and re-streaking
these on fresh agar plates.

Tips for the best results:

 Use only a small amount of inoculum.

 Streak lightly so that you do not gouge the agar.
 Flame the loop after you streak each quadrant.
 Make sure the surface of the plate is free of droplets of condensed moisture.
Figure 7.7 Quadrant Streaking for isolation into pure culture

Procedure

a. Sterilize the inoculating loop in the alcohol burner by putting the loop into the flame until it is
red hot. Allow it to cool.
b. Pick an isolated colony from the agar plate culture and spread it over the first quadrant
(approximately 1/4 of the plate) using close parallel streaks or Insert your loop into the
tube/culture bottle and remove some inoculum. You don’t need a huge chunk.
c. Immediately streak the inoculating loop very gently over a quarter of the plate using a back
and forth motion (see area 1 in the figure 7.6).
d. Flame the loop again and allow it to cool. Going back to the edge of area 1 that you just
streaked, extend the streaks into the second quarter of the plate (area 2).
e. Flame the loop again and allow it to cool. Going back to the area that you just streaked (area
2), extend the streaks into the third quarter of the plate (area 3).
f. Flame the loop again and allow it to cool. Going back to the area that you just streaked (area
3), extend the streaks into the center fourth of the plate (area 4).
g. Flame your loop once more.

The streaked plate is incubated at 37°C for 24 hours. Examine the colonies grown in the plate
carefully. All colonies should have the same general appearance. If there is more than one type of
colony, each type should be streaked again on a separate plate to obtain a pure culture.

2. SPREAD PLATE TECHNIQUE

Spread plate technique is the method of isolation and enumeration of microorganisms in a mixed
culture and distributing it evenly. The technique makes it easier to quantify microbes in a solution.

Principle of Spread Plate Technique

The spread plate technique involves using a sterilized spreader with a smooth surface made of
metal or glass to apply a small amount of microbes suspended in a solution over a plate. The plate
needs to be dry and at room temperature so that the agar can absorb the microbes more readily. A
successful spread plate will have a countable number of isolated microbial colonies evenly distributed
on the plate.

Procedure of Spread Plate Technique

a. Make a dilution series from a sample (Figure 7.7).

b. Pipette out 0.1 ml from the appropriate desired dilution series onto the center of the surface of
an agar plate.
c. Dip the L-shaped glass spreader into alcohol.
d. Flame the glass spreader (hockey stick) over an alcohol burner, let it cool.
e. Spread the sample evenly over the surface of agar using the sterile glass spreader, carefully
rotating the Petri dish underneath at the same time.
f. Incubate the plate at 37°C for 24 hours.
g. Calculate the CFU (Colony Forming Unit) value of the sample. Once you count the colonies,
multiply by the appropriate dilution factor to determine the number of CFU/mL in the original
sample.

Uses of Spread Plate Technique

1. It is used for viable plate counts, in which the total number of colony forming units on a single
plate is enumerated.
2. It is used to calculate the concentration of cells in the tube from which the sample was plated.
3. Spread plating is routinely used in enrichment, selection, and screening experiment

Figure 7.8 Serial dilution procedure

Figure 7.9 Spread plate technique
3. OBSERVATION AND IDENTIFICATION

Procedure:

a. Choose a microbial colony, use the inoculation loop pick bacterial cells and put it into a water
droplet on a microscope slide.
b. Observe cell shape under a microscope (400x magnification) and take a photo.
c. Identify the microorganism group through cell characteristics.
d. Describe the characteristics of selected colony (as in table 7.1).
e. Repeat step (a) to step (d) with a colony of another group.
 VIETNAM NATIONAL UNIVERSITY HCM CITY THE REPORT OF LABWORK ON
UNIVERSITY OF SCIENCE GENERAL BIOLOGY 1
FACULTY OF BIOLOGY - BIOTECHNOLOGY Date: ……………………………

MICROBIAL DIVERSITY

I. INFORMATION OF STUDENT
Stage: …………. (…………….. to …………….) Group: ………………
Full name: ……………………………………….. The student ID number: ………………………
Full name: ……………………………………….. The student ID number: ………………………
Full name: ……………………………………….. The student ID number: ………………………
II. RESULTS
1. Observation and identification

BACTERIUM

Colony Cell

ACTINOMYCETE

Colony Cell
 FUNGUS

Colony Cell

YEAST

Colony Cell

MICROALGA

Colony Cell
Insert images into the blanks
Table 7.1 Morphology of microbial colonies
Morphology Bacterium Actinomycete Fungus Yeast
Circle
Punctiform
Shape Filamentous
Irregular
Rhizoid
Small
Size Medium
Large
Smooth
Glistening
Surface
Rough
Wrinkled
Transparent
Opacity Opaque
Translucent
Flat
Umbonate
Elevation Raised
Convex
Cracteriform
Even
Wavy
Margin Filamentous
Lobate
Curled
Dry
Viscid
Texture
Smooth
Mucoid
White
Creamy-white
Color Yellow
Orange
Green
Use symbol (+) for the characteristics found in microbial colonies
2. Streaking & Spreading

Streak plate Spread plate

Spreading
Volume of water (mL)
Volume of inoculum (µL)
Step
Dilution factor
CFU/plate
Density of microorganisms
(cells/mL)