Lesson 3

The Prokaryotic Cell

Introduction

The provided lecture material offers a comprehensive overview of prokaryotic cells,

with a specific focus on bacteria. It is structured to introduce the fundamental
distinguishing characteristics of prokaryotes and then delve into the specifics of
bacterial morphology and external and internal structures.

Specific Objectives
At the end of the lesson, the students should be able to:
1. Discuss the characteristics that differentiate prokaryotic cells with eukaryotic
ones; and
2. Differentiate the cell wall characteristics of Gram + and Gram – bacteria.

Duration
Lesson 3: The Prokaryotic Cell = 12 hours

Distinguishing Characteristics of Prokaryotes

1. Prokaryotic DNA is not enclosed within a membrane and is one circular

chromosome.
2. Prokaryotic DNA is not associated with histones but other types of proteins.
3. Prokaryotic cell lacks membrane-bound organelles.
4. Prokaryotic cell walls almost always contain the complex polysaccharide
peptidoglycan.
5. Prokaryotic cell often divides by binary fission where the prokaryotic DNA
is copied and the cell splits into two cells.

4.1 SIZE, SHAPE AND ARRANGEMENT OF BACTERIAL CELLS

Bacteria are unicellular and most bacterial species are differentiated by morphology,
chemical composition, nutritional requirements, biochemical activities and source of
energy.

4.1.1 Size
- most bacteria are 0.2 – 2.0 µm in diameter and 2 – 8 µm in length

4.1.2 Shape and Arrangement

- three basic bacterial shapes are coccus, bacillus and spiral

a. Coccus (pl. cocci , meaning berries)

- are usually round but can be oval, elongated or flattened on one side
- types:
1. Diplococci – cocci that remain in pairs after dividing
 2. Streptococci – cocci that divide and remain attached in
chainlike patterns
3. Tetrads – cocci that divide in two planes and remain in
groups of four
4. Sarcinae – cocci that divide in three planes and remain
attached in cubelike groups of eight
5. Staphylococci – cocci that divide in multiple planes and
form grapelike clusters or broad sheets

Figure 12. Cocci Shape and Arrangement of Bacteria

[Link]
and_Prokaryotic_Cell_Anatomy/2%3A_The_Prokaryotic_Cell__Bacteria/2.1%3A_Sizes_Shapes_and_Arrangements_of_Bacteri
a

b. Bacillus (pl. bacilli, meaning little staffs)

- bacilli divide only across their short axis, thus, fewer groupings are
seen compare to cocci and most bacilli appear as single rods
- types:
1. Diplobacilli – appear in pairs after division
2. Streptobacilli – occur in chains
3. Coccobacilli – appear-like a combination of coccus and
bacillus
4. Palisades – the bacilli after cell division bend and therefore
arranged in a palisade, fence-like structure
- bacillus has two meanings in microbiology where one refers to a
bacterial shape while when capitalized and italicized, it refers
to a specific genus like Bacillus anthracis.
.

Figure 13. Bacilli Shape and Arrangement of Bacteria

[Link]

2
 c. Spiral
- bacteria that are never straight and have one or more twists
- types:
1. Vibrio – bacteria that look like curved rods
2. Spirilla – have a helical shape (like corkscrew) and fairly rigid
bodies; uses flagella for movement
3. Spirochetes – another group of spirals are helical and flexible;
uses axial filaments to move contained within the
flexible external sheath

Figure 13. Spiral Shape and Arrangement of Bacteria

[Link]
OvVaw2YuUCB5EWJ56m0Gxx3IeUs&ust=1757242352380000&source=images&cd=vfe&opi=89978449&ved=0C

d. Other Bacterial Shapes

1. Star-shaped cells – example genus Stella
2. Rectangular, flat cells - example is halophilic archaea of the
genus Haloarcula
3. Triangular cells

→ The shape of a bacterium is determined by heredity. Two types of bacterial shapes

based on genetics.
a. Monomorphic – most bacteria maintain a single shape but can be altered by
a number of environmental conditions
b. Pleomorphic – bacteria that have many shapes such as Rhizobium and
Corynebacterium

3
 Figure 14. Summary of Bacterial Shapes and Arrangement
[Link]
4.2 STRUCTURES EXTERNAL TO THE CELL WALL

4.2.1 Glycocalyx
- a viscous or sticky gelatinous polymer that is external to the cell wall and
composed of polysaccharide, polypeptide or both; general term used for
substances that surround the bacterial cells
- types: capsule, slime layer & extracellular polysaccharides (EPS)

a. Capsule
= glycocalyx substance that is organized and is firmly attached to the
cell wall
= capsule protects bacteria and also important in contributing to the
bacterial virulence for it protects the pathogenic bacteria from
phagocytosis.
= a glycocalyx can protect a cell against dehydration as well as its
viscosity may inhibit the movement of nutrients out of the cell
Example: Streptococcus pneumoniae cannot cause pneumonia
and are easily phagocytized when unencapsulated; only
capsulated S. pneumonia causes the disease

Figure 15. Types of Bacterial Glycocalyx found External of the Cell Wall
10.5005/jp/books/12637_3

b. Slime layer
= a glycocalyx that is unorganized and is only loosely attached to the
cell wall.

c. Extracellular Polysaccharide (EPS)

= a glycocalyx made of sugars
= EPS enable a bacterium to attach to various surfaces in its natural
environment in order to survive

4.2.2 Flagella
- long filamentous appendages that propel bacteria
- a semirigid, helical structure that moves the cell by rotating from the basal
body
- Parts:
a. Filament
= the long outermost region with a constant diameter and contains
flagellin (globular protein)
b. Hook

4
 = connects the filament to the basal body
= enables the filament to turn to about 360˚
c. Basal body
= stack of rings with rod that anchor the flagellum to the cell wall and
plasma membrane
= Gram (-) bacteria contain two pair of rings – one pair of rings is
anchored to some portions of the cell wall and another pair of rings
is anchored to the plasma membrane.
= Gram (+) bacteria, only the inner pair is present.

Figure 16. Structure of Bacterial Flagella

[Link]

- 4 Flagella Arrangements:

a. Monotrichous – a single polar flagellum

b. Amphitrichous – a tuft of flagella at each end of the cell
c. Lophotrichous – two or more flagella at on pole of the cell
d. Peritrichous – flagella distributed to the entire body

Figure 17. Flagellar Arrangements in Bacteria

[Link]

5
- the rotation of the basal body of the flagellum is either clockwise or counterclockwise
around its long axis.
- as the flagella rotate, they form a bundle that pushes against the surrounding liquid
and propels the bacterium.

→ Flagellar rotation depends on the cell’s continuous generation of energy

→ Flagellum is effective in guiding bacteria through the environment primarily because
the system for detecting chemicals is linked to the mechanisms that drive the
flagellum.
= located in the cell membrane are clusters of receptors that bind specific
molecules coming from the immediate environment that signals the flagellum
into rotary motion.
= Taxis is the movement of a bacterium towards or away from a particular
stimulus
a. Chemotaxis – stimuli that includes chemicals
b. Phototaxis – light stimuli

→ The flagellar protein called H antigen is useful for distinguishing among serovars
(variations within a species) of gram-negative bacteria.

4.2.3 Axial Filaments

- also known as endoflagella or periplasmic flagella
- a type of internal flagellum that is enclosed in the space between the
cell wall and cell membrane.
- commonly found in Spirochetes

4.2.4 Fimbrae and Pili

- shorter, straighter, and thinner than flagella appendages that are used
mainly for attachment rather that locomotion.
- both fimbrae (s: fimbra) and pili (s: pilus) consist of a protein called
pilin that is arranged helically around a central core

a. Fimbrae
= are small, bristlelike fibers spouting off the surface of many
bacterial cells
= they have an inherent tendency to stick to each other and to
surfaces
= they are responsible for the mutual clinging of cells that leads
to biofilms and other thick aggregates of cells on the surface of
the liquids and for the microbial colonization of inanimate solids
such as rocks and glass
= examples: Escherichia coli
b. Pili
= a long, rigid tubular structure

6
 = true pili have been found only in gram-negative bacteria where
they are utilized in a “mating” process between cells called
conjugation which involves partial transfer of DNA from one
cell to another between compatible gram-negative cells.

Figure 18. Electron micrograph of Salmonella typhi showing flagella and fimbriae
[Link]

4.3 BACTERIAL CELL WALL

- complex and semirigid (can extend when needed) structure

- Major function:

1. Prevent rupturing of bacterial cell when water pressure inside the cell
is greater than the outside environment.
2. Maintains bacterial cell shape
3. Serves as anchorage for flagella
4. Clinically, it contributes to the pathogenic capability of some bacterial
species
5. Site of antibiotic action

4.3.1 Composition and Characteristics

- the bacterial cell wall is composed of a macromolecule peptidoglycan

network called murein
- Murein as a peptidoglycan consists of repeating disaccharide attached
to polypeptides.

= NAG-NAM – are the disaccharide portion of the cell wall, related

to glucose, arranged in alternating pattern linked in

7
 rows of 10 – 65 sugars forming the carbohydrate
“backbone”
- (NAG) is N-acetylglucosamine
- (NAM) is N-acetylmuramic acid (Murus meaning wall)
= Polypeptides – the polypeptide of murein always includes tetrapeptide side
chains (4 amino acids attached to NAM backbone) with alternating D and L
forms of amino acid
- Parallel tetrapeptide side chains may be linked by a peptide
cross-bridge
Note: Penicillin disrupts the peptide cross-bridge

Figure 19. The basic structure of bacterial peptidoglycan and the cell wall structures of Gram-positive
and Gram-negative bacteria.
[Link]
modification/peptidoglycans?srsltid=AfmBOoqKqYN3ySv0PxqN4x5UcAdSk6KMLUh1K2mhNh8Xs1EcfnD0VPFN

A. Gram-Positive Cell Wall

= consists of thick, rigid structure
= contains teichoic acids consists of alcohol (glycerol or ribitol)
and phosphate
= Teichoic acid
Function:
1. Binds and regulates the movement of cations
into and out of the cell due to its negative charge.
2. Assume a role in cell growth, preventing
extensive wall breakdown and cell lysis.
3. Provides much of the wall’s antigenic
specificity (gram + bacteria can then be
identified)
Types:
1. Lipoteichoic Acid
- spans the peptidoglycan layer and is
linked to the plasma membrane

8
 Figure 20. Gram-positive Cell Wall 2. Wall lipoteichoic Acid
[Link]
- linked to the peptidoglycan

B. Gram-negative Cell Walls

= consists of one or a very few layers of peptidoglycan

and an outer membrane
= does NOT contain teichoic acid
= the peptidoglycan is bonded to lipoproteins and is in
the periplasm (a fluid-filled space bet. plasma
membrane and outer membrane)
= since peptidoglycan is thin, the gram – cell wall is
susceptible to mechanical breakage.

Figure 21. Gram-negative Cell Wall

[Link]

= Outer Membrane

- consists of lipopolysaccharides (LPS), lipoproteins,

and phospholipids.

- Functions:
1. Its strong negative charge is an important
factor in evading phagocytosis and the actions of
complement proteins.
2. It also provides barriers to certain antibiotics
digestive enzymes like lysozyme, detergents,
heavy metals, bile salts and certain dyes.

- is also semi-permeable due to the presence of porins.

Porins permit the passage of nucleotides, disaccharides,
peptides, amino acids, vitamin B12 and iron.

Figure 22. Gram-negative Outer Membrane

[Link]

- has LPS component consists of polysaccharide portion (O polysaccharide) and lipid

portion (Lipid A).

O polysaccharide = function as antigens and are useful for distinguishing

species of gram – negative bacteria.
= role is comparable to teichoic acids

Lipid A = referred as endotoxin and is toxic when in host’s the bloodstream or

GI tract; it causes fever and shock.

9
 Figure 23. The basic structure of bacterial peptidoglycan and the cell wall structures of Gram-positive
and Gram-negative bacteria.
[Link]
Hassanzadeh/publication/363043765/figure/fig1/AS:11431281084351259@1663155776754/Types-of-microorganisms-based-
on-cell-wall-composition-a-Gram-positive-and-b_W640.jpg

4.4 STRUCTURES INTERNAL TO THE CELL WALL

4.4.1 Inclusions/ Reserve Deposits

= internal structures where accumulations of certain nutrients when they are
plentiful and use them when the environment is deficient are found.

a. Metachromatic Granules
- collectively known as volutin that represents as reserves of inorganic
phosphate that can be used in the synthesis of ATP.
- characteristic of Corynebacterium diphtheriae which is causative agent and
use as clinical diagnosis of diphtheria.

10
 b. Polysaccharide Granules
- consists of glycogen and starch

c. Lipid Inclusions
- revealed using Sudan dyes
- a common lipid storage material that is unique to bacteria is the polymer poly-
β-hydroxybutyric acid.

Figure 24. A typical prokaryotic cell contains a cell membrane, chromosomal DNA that is concentrated in a
nucleoid, ribosomes, and a cell wall. Some prokaryotic cells may also possess flagella, pili, fimbriae, and capsules.
[Link]
02%3A_Unique_Characteristics_of_Prokaryotic_Cells

d. Sulfur Granules
- sulfur bacteria like Thiobacillus that derive energy by oxidizing sulfur and
sulfur-containing compounds found in these granules.

e. Carboxysomes
- are inclusions that contain the enzyme ribulose 1,5-diphosphate carboxylase.
- Bacteria that use CO2 as their sole source of C require this enzyme for CO2
fixation during photosynthesis.
- Among the bacteria containing carboxysomes are nitrifying bacteria,
cyanobacteria and thiobacilli.

f. Gas Vacuoles
- this are hollow cavities found in many aquatic prokaryotes.
- each vacuole consists of rows of several individual gas vesicles, which are
hollow cylinders covered by proteins.
- Gas vacuoles maintain buoyancy so that the cell can remain at the depth in the
water appropriate for them to receive sufficient amounts of oxygen, light, and
nutrients.

g. Magnetosomes
- this are inclusions of iron oxide (Fe3O4) formed by several Gram negative
bacteria that act like magnets Aquaspirillum magnetotacticum.
- in vitro, magnetosomes can decompose hydrogen peroxide and protect cell
against hydrogen peroxide accumulation.

4.4.2 Endospore

11
 = are dormant or resting structures formed by some bacteria (common to
Gram positive) for survival during adverse environmental conditions.
= the process of endospore formation is called sporulation and the return of an
endospore to its vegetative state is called germination.
= endospores are unique to bacteria are highly durable dehydrated cells with
thick cell walls and additional layers.
= they are formed internal to the bacterial cell membrane and when released in
the environment, they can survive extreme heat, lack of water, and exposure to
many toxic chemicals and radiation.

Figure 25. Bacterial Endospore

[Link]

References/Additional Resources/Readings

Cowan, M.K. and H. Smith. (2019). Microbiology Fundamentals: A Clinical

Approach. 3rd ed. New York: McGraw-Hill Education.

Madigan, M., K, Bender, D. Buckley, W. M. Sattley, and D. Stahl. (2022). Brock

Biology of Microorganisms. 16th ed. United Kingdom: Pearson Education Limiteds

Tortora, G., B. Funke, & C. Case. (2019). Microbiology: An Introduction. 13th ed.
USA: Pearson Education, Inc.

12
Lesson 4

Microbial Laboratory Techniques and

Procedures
Introduction

Microbiology is the study of microscopic organisms, such as bacteria, viruses,

fungi, and protozoa. To effectively study these microbes, a wide range of specialized
laboratory techniques and procedures have been developed. These methods are
fundamental to ensuring the safety and accuracy of research, as well as to the critical
fields of medical diagnostics, food safety, and environmental monitoring. A core
principle in any microbial lab is asepsis—the practice of maintaining a sterile
environment to prevent contamination from unwanted microorganisms, thereby
guaranteeing that experimental results are reliable and pure.

The procedures employed in a microbiology laboratory are diverse, spanning

from the preparation of growth media and sterilization of equipment to the isolation and
identification of specific microbial species. Common techniques include streak plating
to obtain pure cultures from a mixed population, differential staining methods like the
Gram stain to classify bacteria based on their cell wall composition, and the use of
various microscopy techniques to visualize microbes and their structures. These
foundational practices are essential for cultivating, observing, and characterizing
microorganisms, providing the data necessary to understand their behaviors, roles, and
interactions in different environments.

Specific Objectives
At the end of the lesson, the students should be able to:
1. Discuss and understand the basic laboratory techniques and procedures
commonly employed in microbiological work (microscopy, aseptic techniques,
staining, cell, preparation, culture media preparation, sterilization, pure culture
techniques) through laboratory.

Duration
Lesson 3: Microbial Laboratory = 9 hours
Techniques and Procedures

Lesson Proper

Preparation and Staining of Specimens

Although living microorganisms could be directly examined under the light
microscope, they often must be fixed and stained to:
- increase visibility,
- accentuate specific morphological features, and
- preserve them for future study

I. FIXATION
- is the process by which the external and internal structures of cells and
microorganisms are preserved and fixed in position.

13
 - it inactivates enzymes that might disrupt cell morphology and toughens cell
structures so that they do not change during staining and observation.
- a microorganism usually is killed and attached firmly to the microscope slide
during fixation.

- Two Types of Fixation Methods:

1. Heat Fixation – bacterial smears are fixed by gently flame heating an

air-dried film of bacteria.
- it adequately preserves overall morphology but not
structures within cells.

Figure 26. Bacterial Heat Fixation

[Link]

2. Chemical Fixation – chemical fixatives penetrate cells and react with

cellular components, usually proteins and lipids, to render them
inactive, insoluble, and immobile.
- chemical fixation must be used to protect fine cellular
substructure and the morphology of larger, more delicate
microorganisms.
- common fixative features contain such components as ethanol,
acetic acid, mercuric chloride, formaldehyde, and
glutaraldehyde.

II. STAINING
- is a method by which a chemical or a dye is applied to make the specimens
more visible under the microscope.
- advantages of staining:
1. Provides contrast between the microorganism under study and the
background.
2. Allows the study of the cell’s structure such as nucleus, vacuoles, cell
wall and other parts of the microorganism.

- Dyes or Stains
= are salts composed of cations and anions affected by pH.
= all dyes have two common features:
a. They all have chromophore groups, groups with conjugated
double bonds that give the dye its color.
b. They can bind with cells by ionic, covalent, or hydrophobic bonding
(+ charged stains binds with – charged structures).

14
 = Two general types of ionized dyes or stains based on the nature of their
charged group:

1. Basic Dyes/ Stains

- methylene blue, basic fuschin, crystal violet, safranin,
malachite green
- have positively charged groups (usually some form of
pentavalent nitrogen) and generally sold as chloride salts.

→ Basic dyes bind to negatively charged molecules like

nucleic acids and many proteins.
→ Basic dyes/ stains are usually used to bacteria since their
cell surface is negatively charged.

2. Acid Dyes/ Stains

- eosin, rose bengal and acid fuschin
- possess negatively charged groups such as carboxyls
( - COOH) and pentahydroxyls ( - OH)

→ Acid dyes/ stains bind to positively charged cell structures.

= The pH may alter staining effectiveness since the nature and degree of
the charge on the cell components change with pH.
→ Anionic dyes stain best under acidic conditions
→ Basic dyes are most effective at higher pHs

- Staining Techniques

1. Simple Staining
- the simplest type of staining in which a single staining agent is
used.
- the value of simple staining lies in its simplicity and ease of use
- here, the fixed smear is covered with a single staining agent,
washes the excess stain off with water, and blots the slide dry.
- basic or acid dyes are used for simple staining frequently to
determine the size, shape and arrangement of bacteria.
Figure 27. Microscopic view of Bacillus (rod) shaped bacteria simple
stained with crystal violet.
[Link]
eneral_Microbiology_Laboratory_2021_%28Lee%29/04%3A_Staining_Techn
iques/4.01%3A_Introduction_to_Staining

Methylene Blue Carbol fuschin Safranin

Figure 28. Types of Microbial Stains

[Link]

15
 2. Differential Staining
- microorganisms react differently from different staining procedures
due to their differences in physical and chemical make-up which is
the basis of differential staining.

- Types of Differential Staining:

1) Gram Staining
= developed by Danish Physician Christian Gram
= it is the most widely employed staining method in
bacteriology.
= it is a differential staining because it divides bacteria
into two classes – Gram Negative & Gram Positive
Bacteria
= General Steps:
a) Fixation
b) The smear will be stained with the basic dye
crystal violet or the primary stain.
c) Followed by treatment with an iodine solution
functioning as mordant (increases the
Figure 29. Steps in Differential Staining
interaction between the stain and the cell).
[Link] d) Then, the smear is decolorized by washing
staining/
with ethanol or acetone.
e) Finally, the smear is counterstained with a simple, basic dye
different from the crystal violet such as Safranin which gives
pink to red color with gram-negative bacteria.

2) Acid-fast Staining

= Such staining method uses harsher treatment –

the Ziehl-Neelsen Method or heating with a
mixture of basic fushin and phenol.
= In this staining method, once the basic fuschin
has penetrated with the aid of heat and phenol, the
acid-fast cells are not decolorized by an acid-
alcohol wash and thus, remain red (due to high
lipid concentration on the wall).
= Non-acid-fast bacteria are decolorized by acid-
alcohol and when counterstained with methylene
blue remain blue.
= Used in staining Mycobacterium tuberculosis
and M. leprae which are responsible for
tuberculosis and leprosy.

Figure 30. Steps in Acid-Fast Staining

[Link]

16
 3. Structural Staining
- specialized staining methods developed to study specific bacterial
structures in light microscope.

- Types of Structural Staining:

1) Negative Staining
= a technique that reveals the presence of the diffuse capsules
surrounding many bacteria.
= Bacteria are mixed with India ink or Nigrosin Dye
and spread out in a thin film on a slide.
= Bacteria appear as lighter bodies in the midst of blue-
black background since ink and dye particles cannot
penetrate either the bacterial cell or capsule.
= Advantage: little distortion of bacterial shape and the
cell can be counterstained for even greater visibility.
Figure 31. Negatively Stained Bacillus
A) Vegetative Cell; B. Endospore
[Link]
principle-reagents-procedure-and-result/

2) Spore Staining

= Endospore is a dormant and resistant structure capable

of surviving for long periods in an unfavorable
environment formed by bacteria like Bacillus and
Clostridium when faced on harsh environment.
= common spore staining process is known as
Schaeffer-Fulton Procedure:

→ In this procedure, a bacterium with endospore

is heated with malachite green (a very strong
stain that can penetrate endospore).
→Then, the rest of the cell is washed free of dye
with water and is counterstained with safranin.
= this technique yields a green endospore resting
in a pink to red cell.

Figure 32. Spore Staining Procedure

[Link]
[Link] 3) Flagella Staining

= such procedure provide taxonomically valuable

information about the presence and distribution
pattern of flagella.
= however, flagella is a very thin cell appendage
and can only be seen in electron microscope.
= to be seen on light microscope:
→ the thickness of the flagella is increased
by coating them with mordant such as tannic
Figure 33. Flagella Staining acid and potassium alum
[Link]
gella-staining/

17
 → then, they are stained with pararosaniline
(Leifson Method) or basic fuschin (Gray
Method).

III. STERILIZATION

-is the process by which all living cells, viable spores, viruses and viroids are
either destroyed or removed from an object or habitat.
- comes from Latin sterilis, unable to produce offspring or barren

- Disinfection
= is the killing, inhibition or removal of microorganisms that may cause
diseases.
= Primary goal is to reduce pathogens
= Disinfectants – are usually chemical agents that perform disinfection
and normally used only on inanimate objects

*A disinfectant does not necessarily sterilize an object for viable spores and few
microorganisms may remain.

- Sanitation
= the process where the microbial population is reduced to levels that
are considered safe by public health standards.

- Antisepsis
= is the prevention of infection or sepsis especially on host tissue
accomplished by chemical agents called antiseptics that prevent
infection by killing or inhibiting pathogen growth and reduce total
microbial nutrition.

Conditions Influencing the Effectiveness of Antimicrobial Agent Activity:

1) Population size
2) Population composition
3) Concentration or intensity of antimicrobial agent.
4) Duration of exposure
5) Temperature
6) Local environment

A. Use of PHYSICAL METHODS in Control

1) HEAT
- Heating is still one of the most popular ways to destroy microorganisms either
moist or dry heat.
- Moist heat readily kills viruses, bacteria and fungi.
- Exposure to boiling water for 10 minutes is sufficient to destroy bacterial
endospores that may survive hours of boiling.
- Boiling can be used for disinfection of drinking water and objects not harmed
by water but it does not sterilize.
- it is essential to have a precise measure of the heat-killing efficiency:

18
Table 3.1 Appropriate Conditions for Moist Heat Killing
Organism Vegetative Cells Spores
Yeasts 5 mins at 50 - 60˚C 5 mins at 70 - 80˚C
Molds 30 mins at 62˚C 30 mins at 80˚C
Bacteria* 10 mins at 60 - 70˚C 2 to over 800 mins at
100˚C
0.5 – 12 mins at 121˚C
Viruses 30 mins at 60˚C

TDT – shortest time needed to kill all organisms in a microbial suspension at a specific
temperature and under defined conditions.
D value – or known as decimal reduction time which is more precise figure than TDT
-> it is the time required to kill 90% of the microorganisms or spores in a sample
at a specified temperature.
-> the food processing industry makes extensive use of D values

- Moist heat sterilization must be carried out at temperatures above 100˚C in order to
destroy bacterial endospores, and this requires the use of saturated steam under
pressure.
= Steam sterilization is carried out with an autoclave, a device somewhat like
a fancy pressure cooker.
→ Water is boiled to produce steam, which is released through the jacket
and into the autoclave’s chamber.
→The air initially present in the chamber is forced out until the chamber
is filled with saturated steam and the outlets are closed.
→ Hot, saturated steam continues to enter until the chamber reaches the
desired temperature and pressure, usually 121˚C and 15 pounds
of pressure.
→At this temp. saturated steam destroys all vegetative cells and
endospores in a small volume of liquid within 10 to 12 minutes.
→Treatment is continued for about 15 minutes to provide a margin of
safety.

Figure 34. Steam Sterilization

[Link]

19
 = Things to remember when autoclaving:
1) All air should be flush out of the chamber or else it will not reach
121˚C.
2) The chamber should not be packed too tightly so that the steam could
circulate freely and contact everything in the autoclave.
3) When a large volume of liquid needs to be sterilized, extended
sterilization time will be needed. (5 L of water = 70 mins)

= Pasteurization is a process where substances such as milk are treated with

controlled heating at temperatures well below boiling point.

Figure 35. Pasteurization

[Link]

- Milk can be pasteurized in two ways:

→In the older method, the milk is held at 63˚C for 30 minutes.

→Flash Pasteurization
= also called as High-Temperature Short-Term (HTST) Pasteurization
= here, large amount of substances like milk are subjected with quick heating
to about 72˚C for 15 secs, then rapid cooling.

20
→ Ultrahigh-temperature (UHT) sterilization
= milk and milk products are heated at 140 to 150˚C for 1 to 3 seconds

= Dry Heat Sterilization

Principle: (1) Protein denaturation, (2) Oxidative damage, (3) Toxic
effect of elevated electrolyte

a) Flaming - is done to loops and straight-wires in microbiology

labs. Leaving the loop in the flame of a Bunsen burner
or alcohol lamp until it glows red ensures that any
infectious agent gets inactivated.

b) Incineration - burn any organism to ash. It is used to sanitize

medical and other biohazardous waste before it is
discarded with non-hazardous waste.

c) Hot Air Oven - powder and oily substances are sterilized in

hot air oven.
- 160°C is maintained (holding) for one hour
→ Dry heat does not corrode glassware and metal instruments as moist
heat does and can be used to sterilize powders, oils, and similar items.
→ It is suitable for heat sensitive materials like may plastics and rubber
items.
→However, it is still less effective than moist heat for it requires longer
time and higher temperature.

2. LOW TEMPERATURE
- Freezing items at - 20˚C or lower stops microbial growth due to lower
temperature and freezing of water.
- Refrigeration greatly slows microbial growth and reproduction but does not
halt it completely.
- Some microorganism will be killed in low temperature but contaminating
microorganisms do not.
- In fact, freezing is a very good method for long-term storage of microbial
samples when carried out properly, and many laboratories have a lower-
temperature freezer for culture storage at – 30 or – 70˚C.
- Thus, refrigeration is a good technique only for shorter-term storage of food

21
 and other items.

3. FILTRATION

- it is an excellent way to reduce microbial population; rather than directly

destroying contaminating microorganisms, the filter simply removes them.

- Two types of filters:

1) Depth Filters
= consist of fibrous or granular materials that have been
bonded into a thick layer filled with twisting channels
of small diameter
= these are made up of diatomaceous earth, unglazed
porcelain, asbestos and other similar material.

2) Membrane Filters

= have replaced depth filters for many purposes.

= membrane filters are circular filters which are porous
membranes, a little over 0.1 mm thick, made of
cellulose acetate, cellulose nitrate, polycarbonate,
polyvinylidene fluoride or any synthetic materials.
= nowadays, membrane filters of 0.2 µm in diameter of
pores are available and are able to filter vegetative cells
but not viruses.
= membrane filters remove microorganisms by screening
them out much as a sieve separates large sand particles
from small ones.

→ Air also can be sterilized by filtration such as surgical masks and cotton plugs on
culture vessels that let air in but keep microorganisms out.
→ Laminar flow biological safety cabinets employing high-efficiency particulate
air (HEPA) filters, which remove 99.97% of 0.3 µm particles, are one of the most
important air filtration systems.

4. RADIATION
- UV radiation kills all kinds of microorganisms due to its short wavelength
(approx. 10 to 400 nm) and high energy.
- The most lethal UV radiation has the wavelength of 260 nm, the wavelength
most effectively absorbed by DNA.

- Two types of radiation used:

1) Ultraviolet radiation – around 260nm; does not penetrate dirt films,
glass, water thus only used and put on ceilings of rooms or in
biological safety cabinets to sterilize the air and any exposed
surface.

22
 2) Ionizing radiation – excellent sterilizing agent and penetrates deep
into objects.

- it will destroy bacterial endospores and

vegetative cells, both prokaryotic and
eukaryotic but not viruses.

- Gamma radiation from cobalt 60 is used in cold

sterilization of antibiotics, hormones, sutures,
and plastic disposable supplies such as syringes.

- Gamma radiation or irradiation is also now used

to “pasteurize” meat and other food.

B. Use of CHEMICAL METHODS in Control

Chemical agents usually act as disinfectants because they cannot readily destroy
bacterial endospores. Disinfectant effectiveness depends on concentration, treatment
duration, temperature and presence of organic material.

➔ Phenolics and alcohols = are popular disinfectants that act by denaturing proteins
and disrupting cell membranes. Lysol is a commercial phenol in the market.
= alcohols are bactericidal and fungicidal but not sporicidal. A 10 to 15
minutes soaking is sufficient to disinfect thermometers and small instruments.

➔Halogens (iodine and chlorine) = these substances kill by oxidizing cellular

constituents. Cell proteins may also be iodinated.
= Iodine is applied as a tincture or iodophor.
= Chlorine may be added to water as a gas,
hypochlorite, or an organic chlorine derivative.

➔ Heavy metals = tend to be bacteriostatic agents. They are employed

in specialized situations such as the use of silver nitrate
in the eyes of newborn infants and copper sulfate in lakes
and pools.

➔Cationic detergents = are often used as disinfectants and antiseptics; they

disrupt membranes and denature proteins.

➔Aldehydes = such as formaldehyde and gluraldehyde can sterilize

as well as disinfects because the kill spores.

➔Ethylene oxide gas = penetrates plastic wrapping material and destroys all
life forms by reacting with proteins. It is used to sterilize
packaged, heat-sensitive materials.

23
IV. CULTURE MEDIA PREPARATION

Culture Medium – is a solid or liquid preparation used to grow, transport, and store
microorganisms. The medium must contain all the nutrients the microorganism
requires for growth.

- Specialized media are essential in the isolation

and identification of microorganisms, the
testing of antibiotic sensitivities, water and
food analysis, industrial microbiology, and
others. A medium can either be synthetic or
complex.

1) Synthetic or Defined Media

- is a relatively simple medium containing CO2 as a C source, nitrate or
ammonia as N source, sulfate, phosphate and a variety of minerals and
widely used in many research laboratories.

2) Complex Media
- media that contain some ingredients of unknown chemical
composition.
- commonly used complex media are Nutrient Broth, Tryptic Soy Broth,
and MacConkey Agar.
- contains undefined components like:

a. Peptone – protein hydrolysates prepared by partial proteolytic

digestion of meat, casein, soya meal, gelatin and
other protein sources.
- they serve as sources of C, N and energy.

b. Meat Extract – such as beef extract which contains amino acids,

peptides, nucleotides, organic acids, vitamins and minerals.

c. Yeast Extract – excellent source of B vitamins as well as N and

C compound

→ If solid medium is needed for surface cultivation of microorganisms, liquid media

can be solidified with the addition of 1.0 to 2.0% agar; most commonly 1.5% is used.

Agar – is a sulfated polymer composed mainly of galactose and glucoronic acid

that is extracted from red algae.
- agar is an excellent hardening agent because most microorganism
cannot degrade it and after melting, it can be cooled to about 40˚ to 42˚C
before solidifying and will not melt again until the temperature rises to
about 90˚C.

24
Types of Media:

1) General Purpose Media – support the growth

of many microorganisms regardless of
what they are such as tryptic soy broth
and tryptic soy agar.

2) Enriched Media – provide special nutrients and environmental conditions that

would favor the growth of the desired microorganism and not of the other type.
Example is blood agar since it is enriched with RBC.

3) Selective Media – favors the growth of particular microorganism while inhibiting

other microorganisms.
- bile salts and dyes such as basic fuschin and crystal violet favor the
growth of gram - bacteria by inhibiting the growth of gram + bacteria.
- Examples: Endo agar, Eosin methylene blue (EMB) agar, and
MacCOnkey agar (has added bile salts) which are widely used for
the detection of E. coli

Figure 36. MacConkey Agar in Gram-Negative Bacteria

[Link]

4) Differential Media – are media that distinguish between different groups of bacteria
and even permit tentative identification of microorganisms based on their
biological characteristics.
- Blood agar is also a differential media since it can distinguish between
hemolytic and non-hemolytic bacteria. Hemolytic bacteria like streptococci and
staphylococci isolated from throats produce clear zones around their colonies
because of RBC destruction.

25
 Figure 37. Blood Agar as Differential Agar
[Link]

V. ISOLATION OF PURE CULTURE

Pure Culture – a population of cells (colony) arising from a single cell or one type of
microorganism.

Techniques in Isolating a Pure Culture:

1) Streak Plate Method – in which a loopful of culture is streaked on the

surface edge of an agar plate in one of several patterns.

Figure 38. Streak Plate Method on Agar

[Link]

2) Spread Plate Method – uses a sterile bent-glass rod (L – glass rod) to spread
thoroughly on the agar medium a small volume of dilute microbial
mixture placed at the center of the agar plate.

26
 Figure 39. Successive Serial Dilution Method
[Link]

Figure 40. Spread and Pour Plate Method

[Link]

3) Pour Plate Method – here, the original sample is diluted several

times to reduce the microbial population sufficiently to obtain
separate colonies when plating. Then, small volumes of several
diluted samples are mixed with liquid agar that has been cooled
to about 45˚C, and the mixture is directly poured immediately
into sterile culture dishes.

References/Additional Resources/Readings

Cowan, M.K. and H. Smith. (2019). Microbiology Fundamentals: A Clinical

Approach. 3rd ed. New York: McGraw-Hill Education.

27
Madigan, M., K, Bender, D. Buckley, W. M. Sattley, and D. Stahl. (2022). Brock
Biology of Microorganisms. 16th ed. United Kingdom: Pearson Education Limiteds

Tortora, G., B. Funke, & C. Case. (2019). Microbiology: An Introduction. 13th ed.
USA: Pearson Education, Inc.

28
Activity Sheet
ACTIVITY 1

Name: Score:
Course/Year/Section: Date:

COMMON LABORATORY PROCEDURES & TECHNIQUES

(STERILIZATION)

I. Objectives:
1. To learn common and useful laboratory procedures/manipulations.
2. To appreciate the need to observe proper laboratory techniques to avoid
accidents, contamination and achieve maximum efficiency.

II. Materials:
Test Tube Cotton Medical Gauze
Autoclavable plastics
Erlenmeyer flasks Petri plates Stirring rod
Pipettes New or Used Bond papers

III. Procedure:

A. Preparation of Cotton Plugs for Test tubes and Erlenmeyer flasks

1. Get an adequate amount of cotton and mold it enough to fit the mouth
of a test tube.
2. A popping sound should be heard when the molded cotton was
remove from the mouth of the test tube to ensure that the correct
amount of the plug is obtained.
3. Get around 5 to 6 inches of medical gauze and fold it to half to make
a square shape. Put the molded cotton at the center.

A B

C D

Top view

4. Tie point A and D of the medical gauze over the molded cotton. Do
the same with B and C. Repeat tying A and D over the tied B and
C. Do the same with B and C over the tied A and D.
5. Check again for the popping sound.
6. Put all the test tubes with cotton plugs on an autoclavable plastic and
tie using rubber bands. Set aside for batch autoclaving.
7. Repeat the entire procedure (1 – 6) for the Erlenmeyer flask but use
more cotton for its plug.

29
 *If test tubes to be used have screw cap, rubber stopper or plastic caps,
cover or screw them loosely.

B. Preparation of Petri Plates for Sterilization

1. Get a piece of new or used bond paper.
2. Put the Petri plate at the center of the bond paper. For used paper, put
the Petri plate on the side without any prints.
3. Fold the sides of the paper over the Petri plate.

Petri
Plate

4. Once folded, fold the remaining side into a triangular shape and then,
fold it underneath the plate. Do the same with the other side.

Petri Petri
Plate Plate

5. Stack the covered Petri plates inside the autoclavable plastics and tie
using rubber bands. Set aside for batch autoclaving.

C. Preparation of Stirring Rod and Pipettes for Sterilization.

1. Get a piece of new or used bond paper. Cover the entire stirring rod
or pipettes.
2. Label which is the stirring rod and which is the pipette.
3. Label further which is the upper portion of the pipette and which is
the tip for easy identification and prevent further contamination.

4. Put the covered and labeled stirring rod and pipettes inside the
autoclavable plastics and tie using rubber bands. Set aside for
batch autoclaving.
`
D. Steam Sterilization or Autoclaving using Steam
1. Put the prepared test tubes and Erlenmeyer flasks with cotton plugs,
covered Petri plates, stirring rods and pipettes inside the
autoclave equipment. However, make sure that the equipment
should not be too crowded. Put distilled water inside the
autoclave.
2. Depending on what type of autoclave equipment, the methods will
quite vary. If the equipment is a top loading autoclave, set the
temperature at 121ᴼC and screw open the exhaust valve then
wait for rigorous escape of steam before screwing the exhaust
valve closed.

30
 3. Wait for the reading on the pressure gauge to be at 15 psi before
starting the timer for 15 to 30 minutes.
4. After 15 to 30 minutes, open the exhaustion valve to release the
steam used for sterilization until the pressure gauge reads
ZERO psi before opening the autoclave door.

PRECAUTION!!!

Do not open the autoclave door unless the pressure gauge reads ZERO psi to
prevent explosion and grave injuries on the body.

5. Let the temperature drop before getting the equipment inside to

prevent skin burns.

Attached pictures of your outputs below.

Answer the following comprehensively.

1. Why are test tubes and Erlenmeyer flasks covered with cotton plugs and not other
materials?

2. Why are test tubes covered loosely with screw caps or rubber stoppers?

31
3. Why are Petri plates covered with used paper on the unprinted side?

4. What are the reasons why the microbiology laboratory equipment should not
be too crowded inside the autoclave machine?

5. What are the reasons behind the use of 121ᴼC at 15 psi for 15 to 30 minutes in
autoclaving?

6. Give and discuss other ways of sterilizing microbial laboratory apparatus and
equipment.

32
 ACTIVITY 2

Name: Score:
Course/Year/Section: Date:

COMMON LABORATORY PROCEDURES & TECHNIQUES

(CULTURE MEDIA)

I. Objective:
1. Explain the principles employed in the preparation of commonly used
culture media
2. Properly prepare commonly used culture media
3. Identify the different types of culture media

II. Materials:
Dehydrated media Analytical balance Spatula
Distilled water Petri dishes
Autoclave E-flasks with plug
Hotplate with stirrer Aluminum foil

III. Procedure:
1. Rinse all glassware with distilled water and make sure that all vessels are
clean.
2. Calculate the desired volume of distilled water. Approximately 15mL of
culture media is needed on each medium sized test tube and 20mL of
culture media for petri plate. In case of other sizes of test tube and Petri
plat, fill up to half of the test tube and of the Petri plates.
3. For a 100mL Nutrient Agar (NA), calculate the desired amount of
dehydrated media by this simple formula:

𝑥 28𝑔 𝑜𝑓 𝑁𝐴
=
100𝑚𝐿 𝑜𝑓 𝑤𝑎𝑡𝑒𝑟 1000𝑚𝐿 𝑜𝑓 𝑤𝑎𝑡𝑒𝑟

4. After determining the amount of NA powder, open the culture medium

container in a cool dry place. (Avoid inhaling the powder and
prolonged skin contact).
5. Weigh the powder quickly and accurately using analytical balance. Reclose
the container as soon as possible.
6. On an Erlenmeyer flask, pour half the required volume of distilled water in
the flask, and then pour the weighed quantity of medium and mix
briskly for a few minutes. Pour the rest of the distilled water down the
sides of the vessel to wash any adherent medium back into solution.
7. Media containing agar should be heated before autoclaving. Bring the
medium to boil using the hot plate, stirring constantly without burning.
The resulting solution should be clear, lightly yellow with little or no
undissolved materials
8. Cover the mouth of the flask tightly with a cotton plug wrapped with
aluminum foil and finally sealed with an autoclave indicator tape.

33
 9. Sterilize the solution in the autoclave at 121˚C for 15 to 30 minutes under
the supervision of your laboratory instructor.
10. After sterilization, cool the culture media to about 50˚C or lukewarm
making sure that the media should not solidify.
11. Aseptically dispense the culture media on the Petri dishes. Pour about 20
mL per Petri dish. Allow cooling and solidifying at room temperature
then turn the Petri plate upside down.
12. For slants, aseptically dispense the culture media to about half the volume
of the test tube. Then, slant the test tubes by laying it down
horizontally.
13. Agar plates and slants should be stored at 2 - 8˚C in sealed containers
away from sunlight to avoid loss of moisture. DO NOT FREEZE.

Worksheet. Enumerate and describe the following culture media and differentiate
them according to classification and use.

Culture Media Description Classification Use

1. Nutrient Agar
2. Nutrient Broth
3. Tryptic Soy Agar
4. Blood Agar
5. Chocolate Agar
6. MacConkey
Agar (MAC)
7. Eosin Methylene
Blue (EMB)
8. Mannitol Salt
Agar (MSA)
9. Thayer-Martin
Agar
10. Sabouraud’s
Dextrose Agar
11. Hektoen Enteric
Agar
12. Lowenstein-
Jensen Medium
(LJ)
13. Mueller-Hinton
Agar (MHA)
14. Cetrimide Agar
15. Salmonella-
Shigella Agar
(SSA)
16. Triple Sugar
Iron Agar

34
 ACTIVITY 3

Name: Score:
Course/Year/Section: Date:

CULTIVATION OF MICROORGANISMS

I. Objectives:
1. Demonstrate the proper technique in handling culture tubes
2. Demonstrate the proper technique in plating culture
3. Enumerate and explain the different ways of inhibiting microbial growth.

II. Materials:

Plate Culture of Bacteria Alcohol lamp Hockey stick or Paper clips

NA Petri plates Distilled water Analytical balance
NA Slants Salt Inoculating loop and needle

III. Procedure:

A. Preparation for Serial Dilution

1. Calculate the amount of salt to be used and at it on 100mL sterile water.
2. Dispense 9mL of the 0.9% NaCl Solution on five test tubes with screw
caps. Label each from 1 to 5.
3. Mix the colony of bacteria from the prepared plate culture into 1mL of 0.9%
NaCl Solution.
4. Add the 1mL bacterial suspension to Test Tube #1, cover it with a screw
cap and then shake vigorously.
5. Get 1 mL of bacterial suspension from Test Tube #1 and dispense it to Test
Tube #2, cover and shake.
6. Repeat procedure to Test Tube #3, 4 and [Link] aside for later use.

B. Pour Plate Method

1. Liquefy a prepared slant on a hot water bath at about 50˚C.
2. Aseptically, add 1mL of bacterial suspension from Test Tube #5 to the
liquefied slant and mix well. Do not forget heating the mouths of all
test tubes before and after opening.
3. Get a sterilized Petri plate and pour the mixture on it.
4. When the agar solidifies, incubate the plate for 24 hours at 37˚C.

C. Spread Plate Method

1. Get 1mL of bacterial suspension from Test Tube #5 and put it on the center
of the sterilized prepoured, solidified agar medium Petri plate.
2. Heat the hockey stick or formed paper clip then allow the stick to cool.
3. Using the stick, spread the inoculum uniformly over the medium.
4. Incubate the plate for 24 hours at 37˚C.

D. Streak Plate Method

1. Heat the inoculating loop over direct flame until it grows red.

35
 2. Allow the loop to cool before getting a colony of bacteria from a prepared
culture medium.
3. Hold the new culture media with your left hand in a way that you will be
able to use your left thumb to open the lid.
4. With your right hand, inoculate the loopful of microorganisms using streak
technique on one side of the plate.
5. Remove the loop and close the plate.
6. Turn the plate by a quarter.
7. Heat the loop again and allow it to cool before streaking again into the
plate.
8. Do another streak.
9. Repeat this procedure until you have three streaks. Make sure that the last
streak does not overlap with the first one.
10. Properly wrap the plate, invert and incubate for 24 hours at 37˚C.
11. After incubation, observe the characteristics of the bacterial colonies that
formed. Draw and describe your observation.

E. Nutrient Agar Slants

1. Heat the inoculating loop over direct flame until it grows red.
2. Allow the loop to cool before getting a colony of bacteria from a prepared
culture medium.
3. Hold the new culture media with your left hand, and using the fingers of
your right hand, clip the cotton plug to remove it.
4. With your right hand, inoculate the picked colony by streaking it into the
slant.
5. Remove the needle and return the cotton plug.
6. Repeat this procedure on the remaining test tubes.
7. Place the tube in a rack and incubate for 24 hours at 37˚C.
8. After incubation, observe the characteristics of the bacterial colonies that
formed. Draw and describe your observation.

Observation and Result

1. Draw and describe your observation on pour plate, spread and streak plate method.

Top
View

Side
View

Pour Plate Method Spread Plate Method Streak Plate Method

36
2. Draw and describe your observation on Nutrient Agar Slants

37
Learner’s Feedback Form

Name of Student:
Program :
Year Level : Section/Major :
Faculty :
Schedule :

Learning Module: Number: _________Title:

How do you feel about the topic or concept presented?

□ I completely get it. □ I’m struggling.
□ I’ve almost got it. □ I’m lost.

In what particular portion of this learning packet, you feel that you are struggling or
lost?
_____________________________________________________________________
_____________________________________________________________________
____________________________________________________________________

Did you raise your concern to your instructor? □ Yes □ No

If Yes, what did he/she do to help you?

_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________

If No, state your reason.

_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________

To further improve this learning packet, what part do you think should be enhanced?
_____________________________________________________________________
_____________________________________________________________________
____________________________________________________________________

How do you want it to be enhanced?

_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________

NOTE: This is an essential part of course module. This must be submitted to the subject teacher
(within the 1st week of the class).

38