Microbiology and Parasitology

Laboratory

Lab Lesson 1 The Microbiology Laboratory

Like other laboratories, microbiology laboratory utilizes a number of apparatus and
equipment pertinent to the nature of its discourse. Without these tools experiments and
microbiological studies employing techniques like isolation of microbes and culturing,
sterilization, media preparation and other protocols that could not be done.
Most of the common glass wares used in the other laboratories are useful as culture
tubes and measurement apparatus. Various types of culture media are utilized to support the
growth of most and those desired microorganisms.
Sterile techniques are always utilized in the laboratory. These techniques are used in
the preparation of glass wares, apparatuses, and culture media so as to prevent the spread of
microorganisms and contamination of materials and cultures.
Familiarity of the apparatus, glass wares, equipment, culture media, and sterile
techniques is crucial before any manipulation is undertaken.

A. LABORATORY MATERIALS AND APPARATUSES

Test tube a common reaction vessel used in chemistry laboratory finds useful principally for
growing microorganisms. It is somewhat analogous to the growing of animals in a cage or
plant on a pot. This piece of glass tube is usually stopper with a plug of cotton. There are
several types of test tubes utilized in culturing microorganisms.

1. Durham tube- a small test tube placed inverted inside a large test tube. This serves to
collect and makes visible any gas released by microorganisms during their growth.
2. Smith tube- another piece of apparatus similar to a test tube that measures the quantity
of gas released during microbial metabolism.
3. Screw cap test tube- a test tube threaded at the top and a plastic cap screwed on it.

Petri dishes are made up of glass or plastic and provide a large place in which to grow
microorganisms in artificial cultures. These were first utilized by Hans Petri using agar
based culture medium. Each petri dish is consists of 2 disc-like parts that look alike except
that one is slightly larger and fits over the smaller forming a cap.

Pipets/pipetors are essentially a piece of glass tubing that has been calibrated and drawn out
to a capillary-type tip. At the other end the pipette has a mouthpiece where the suction bulb
is applied to draw the fluid into the pipette. This is used to deliver specified quantities of
fluid from one container to another.

Indicators are substances widely used in microbiological work, which by some visible change,
such as a change of color indicates the condition of a solution as to the presence of free
acid, alkali or other substances. All indicators change color depending upon the pH in which
they are dissolved.

Buffers are salts of either weak acids or weak bases that tend to resist a rapid change in pH
from the pH at which the medium was originally set.

_____________________________________________________________________________________
Lab Lesson 1: The Microbiology Laboratory
1
 Microbiology and Parasitology
Laboratory
Bacteriological incubator is very similar to an ordinary oven that is thermostatically controlled
to maintain a constant temperature.
Bacteriological Sterilizers
a) Autoclave is a large pressure cooker type device on which the boiling temperature of water
is increased by an increase in pressure
b) Oven is used in dry sterilization. Glassware and other inert objects are sterilized in this
fashion.
c) Cellulose membrane filters prevent the passage of all bacteria and larger microorganisms
of fluid passing through them. The microfiltration set-up includes a membrane filter funnel,
suction flask, rubber tube and suction pump

Inoculating Loop and Needle

These devices are consist of a needle to which is attached a piece of wire made of some
inert metal such as nichrome or platinum. Inoculating loops and needles are often used for
purpose of transferring microbial growth from one container to another without introducing
contaminants. The wire is sterilized by flaming to red heat then allowed to cool for about 10-
15 seconds.

Alcohol Lamp
A lamp fuelled with denatured alcohol use in sterilizing inoculating loops and needles
through direct flaming. Combustion of alcohol produces clean blue flame.

Bent Glass Rod

A piece of glass tube curve into L-shape which is used as spreader of bacterial sample in an
agar plate employing spread plate technique in isolating pure culture of microorganisms
especially bacteria.

Bacteriological Slides
Several types of slides are used in microbiology. The plain glass slide is the most
common type of slide used in making temporary and permanent microbial smears. The
concave slide has a hollowed-out portion in the center. The slide is used for observing
living microorganisms. Growing a micro culture of fungi uses deep well slide. The entire
upper surface is frosted and the depression is cylindrical rather than concave.

B. CULTURE MEDIA
Growing microorganisms in vitro requires mixtures of nutrients known as culture media.
Media may serve many different purposes in microbial work.
Media are classified based on their composition, physical state, and purpose.

A) According to composition:
 Chemically-defined media are those in which the exact chemical compositions are
known. For the growth of autotrophic microbes and microbial assays. Table 1-1
shows an example of a chemically-defined medium.
 Complex media are natural media like milk, blood and other nutrients derived from
animal, plant and yeast extracts (table 1-2). Exact compositions vary depending on
the source.

B) According to purpose:
_____________________________________________________________________________________
Lab Lesson 1: The Microbiology Laboratory
2
 Microbiology and Parasitology
Laboratory
 General-purpose media will support a large number of organisms.
 Selective media provide nutrients that enhance the growth and predominance of a
particular type of microbe while unwanted types are suppressed.
 Differential media allow distinctive growth of various wanted microbes making them
easy to identify.

Table 1-1 Chemically-defined medium for growing a typical

chemoheterotrophs, such as Escherichia coli.
Constituent Amount
Glucose 5.0 g
Ammonium phosphate, monobasic 1.0 g
(NH4H2PO4)
Sodium chloride (NaCl) 5.0 g
Magnesium sulfate (MgSO4.7H2O) 0.2 g
Potassium phosphate (K2HPO4) 1.0 g
Water 1 liter

 Reducing media support growth of obligate anaerobes. Sodium thioglycolate

depletes the medium of oxygen by combining with dissolved oxygen in the medium.
 Enrichment media are media designed to increase numbers of wanted microbes in a
detectable level. This is used to grow fastidious microbes.

Table 1-2 A complex medium for the growth of heterotrophic

bacteria.
Constituent Amount
peptone 5.0 g
beef extract 3.0 g
Sodium chloride 8.0 g
Agar 15.0 g
Water 1 liter

C) According to state:
 Solid media have agar-agar or gelatin added to them as solidifying agent. They are
used for the study of colony morphology, isolation of pure cultures and prolong
storage of pure cultures.
 Semisolid media have lesser amount of agar, thus they have a jelly-like consistency.
These are used in sugar utilization and motility tests.
 Broths are liquid media lacking solidifying agents.

_____________________________________________________________________________________
Lab Lesson 1: The Microbiology Laboratory
3
 Microbiology and Parasitology
Laboratory

ACTIVITIES

A. LABORATORY APPARATUS

Objective
At the end of the exercise the student must be able to identify the common apparatuses
and equipment used in microbiology laboratory and correctly state their functions.

Materials
test tubes of various designs indicators
petri dish buffers
pipets and pipetors incubator
autoclave oven
membrane filters slides of various types
bent glass rod alcohol lamp
inoculating loops and needles

Procedure
1. Examine the different apparatus/equipment as describe earlier. Draw each
apparatus/equipment.
2. Familiarize the functions/uses of the different equipment.

B. CULTURE MEDIA

Objective
At the end of the exercise the student must be able to classify the different types of
media used in culturing microorganisms and state their purposes.

Materials
Culture media

Procedure
1. Observed the different types of media provided by your instructor.
2. Examine the ingredients of each culture medium and classify whether it is a
chemically-defined or complex medium. Also note the uses/purposes stated in the
label. Analyze it to determine whether the medium is general purpose, selective or
differential. Fill-in the data in table 1-1.
3. Examine closely the ingredients of the following culture media: Nutrient Broth,
Lactose Broth, Potato Dextrose Agar, Simmon’s Citrate Agar and Trypticase Soy
Agar. Determine which ingredient is the primary carbon source (carbohydrates) and
nitrogen (proteins). Complete Table 1-2.

_____________________________________________________________________________________
Lab Lesson 1: The Microbiology Laboratory
4
 Microbiology and Parasitology
Laboratory

_____________________________________________________________________________________
Lab Lesson 1: The Microbiology Laboratory
5
 Microbiology and Parasitology
Laboratory
Name: _____________________________ Score: ____________________
Lab Schedule: _______________________ Term/Date: ___________________

The Microbiology Laboratory Lab Report 1

Results and Observation Attendance
prelab: _______________
postlab: ______________
A-1. LABORATORY MATERIALS AND APPARATUSES

------------------------------------------------------ ------------------------------------------------------
Durham tube Smith tube

_____________________________________________________________________________________
Lab Lesson 1: The Microbiology Laboratory
6
 Microbiology and Parasitology
Laboratory

------------------------------------------------------ ------------------------------------------------------
Screw-cap test tube Petri dish

------------------------------------------------------ ------------------------------------------------------
Pipets and pipettors Bacteriological incubator

_____________________________________________________________________________________
Lab Lesson 1: The Microbiology Laboratory
7
 Microbiology and Parasitology
Laboratory

------------------------------------------------------ ------------------------------------------------------
Autoclave Oven

--------------------------------------
Cellulose membrane filter

------------------------------------------------------------------------------------------------------- --------------------------------------
Microfiltration set- up Alcohol lamp

------------------------------------------------------
Plane glass slide

-------------------------------------------------------
Concave slide

_____________________________________________________________________________________
Lab Lesson 1: The Microbiology Laboratory
8
 Microbiology and Parasitology
Laboratory

------------------------------- ------------------------------- ------------------------------------------------------

Inoculating needle Inoculating needle Deep-well slide

_____________________________________________________________________________________
Lab Lesson 1: The Microbiology Laboratory
9
 Microbiology and Parasitology
Laboratory
A-2. Match the functions with one of the following equipment and apparatuses.

____ autoclave ____ durham tube ____ petri dish

____ buffer ____ incubator ____ plain glass slide
____ cellulose membrane filter ____ indicators ____ screw cap test tube
____ concave slide ____ smith tube ____ pipette
____ inoculating loop and needle ____ deep well slide ____ oven

a) collect any gas produced during microbial metabolism

b) culture tube with plastic screw cap
c) deliver specified quantities of fluid from container to another
d) designed for transferring microbial growth
e) determine the pH of solutions
f) for observation of living microorganisms
g) fungal microculture
h) maintains a preferred constant temperature for growing microorganisms
i) preparation of bacterial smears
j) pressure cooker type device for wet sterilization
k) prevents hazardous change in pH
l) provides larger place to grow microorganisms in artificial medium
m) sterilizes glass and inert objects
n) sterilizes heat sensitive solutions

B. CULTURE MEDIA
Classify the following media as chemically-defined (CDM), complex (CXM), general
purpose (GPM), selective (SM) and differential (DM) and describe their uses.

Table 1-1. Classification of culture media.

Type
Media According to According
Uses
composition to purpose
Bacillus cereus
Selective Agar
Base
Blood Agar
Base
Brilliant Green
Lactose Broth
Desoxycholate
Agar
Endo Agar
Base
Endo MF Broth
Eosin
Methylene Blue
Agar (Levine)

_____________________________________________________________________________________
Lab Lesson 1: The Microbiology Laboratory
10
 Microbiology and Parasitology
Laboratory
Type
Media According to According
Uses
composition to purpose
E.C. Medium
King B Medium

Lactose Broth

Mannitol Nitrate
Motility Agar
Mannitol Salt
Agar
McConkey Agar

Methyl Red
Voges
Proskauer Broth
(MRVP)
Mueller Hilton
Agar
Nutrient Agar

Nutrient Broth

Phenol Red
Broth Base
Potato Dextrose
Agar
Sabouraud
Dextrose Agar
Salmonella-
Shigella Agar
Simmon’s
Citrate Agar
Staphylococcus
Agar NO110
Streptococcus
Selection Agar
Thioglycollate
Fluid Medium
Triple Sugar
Iron Agar
Trypticase Soy
Agar
Urea Indol
Broth

_____________________________________________________________________________________
Lab Lesson 1: The Microbiology Laboratory
11
 Microbiology and Parasitology
Laboratory

Table 1-2. Comparison of carbon and nitrogen sources of selected culture media.
Culture Media Carbon (energy) Source Nitrogen (protein) Source
1. Nutrient Broth
2. Lactose Broth
3. Potato Dextrose Agar
4. Simmon’s Citrate Agar
5. Trypticase Soy Agar

Match the following media with their appropriate uses. Write the letters on the blanks before
the number.

_____ Differential media a) culture of fastidious microbes

_____ Enrichment media b) isolation of desired microbes
_____ General-purpose media c) culture of obligate anaerobes
_____ Reducing media d) culture of various types of microbes
_____ Selective media e) distinguish growth of various desired microbes

_____________________________________________________________________________________
Lab Lesson 1: The Microbiology Laboratory
12