Microbial Techniques

I- Sterilization:
■ A)Physical methods
■ B)Chemical methods
■ C)Radiation methods
II-Staining Techniques
■ Simple Stains
■ Differential Stains
■ Structural Stains
Sterilization is a Process by which an object is made free of microbes.
Microbiocidal: here microbes are killed.
Microbiostasis: here microbes are prevented from multiplication.
Sanitization: Process of reducing the level of microbial flora to safe level for public health.
Germicide: Agent capable of killing vegetative cells.
Sporicide: Agent capable of killing spores.

I Physical methods:
a. Autoclave
b. Hot air oven
c. Laminar air flow
d. Seitz filter
e. Sintered filter
f. Membrane filter

1.Seitz filter/Asbestos filter: It is a circular disc thickness of 2-6mm composed of Asbestos fibers.
Disc is fitted to filter assembly made up of stainless steel, then pressure is applied for effective filtration.
Filters are soft and easily get damaged.
2.Sintered filter: It is prepared by heating glass powder, fusing them to a disc form in a suitable mold
then sealed to a funnel.
3.Membrane filter/Ultra filter: It is a circular porous membrane of 0.1 mm thickness made up of
cellulose acetate and polycarbonate. Millipore filters 8-19µm.

II Chemical methods: Chemical methods are most efficient and popular sterilization methods.

1. Alcohol: 70% Ethanol hand disinfectant, germicide kills vegetative cells.

Methanol kills fungal spores but toxic to eyes
Iso propanol is more bactericidal.
Mode of action: Membrane disruption and protein denaturants.

2. Aldehydes: Formaldehyde: disinfectant sporicide lethal on viruses.

Glutaraldehyde: fungicide effect on viruses.
Denatures proteins, nucleic acids.

3. Phenols: Poisonous disinfectants.

Joseph Lister father of Antiseptic surgery used to treat post operational wounds.
Denatures proteins and enzymes.

4. Halogens:
Disinfectants antiseptic oxidizing agents.
Chlorine, Iodine

5. Gaseous agents:
Ethylene oxide- it is a liquid below 10oc then vaporizes highly inflammable, toxic, irritating.
Alkylating agent replaces H with alkyl group which inactivates enzymes.
β Propiolactone-more effective irritating to eyes. Kills bacteria, fungi, viruses and spores.
Other agents-
Metallic salts like mercuric chloride
Dyes- aniline, acridine dyes
Detergents-Tween20
Sodium hypo chloride, H2O2, chloroform.

III Radiation methods: Radiation is the energy transmitted through space in variety of forms.

I Non ionizing Radiation: longer wavelength, less energy, less penetrating power.

UV rays- 200-400nm

Potent Bactericidal activity on vegetative cells & spores. Have less penetrating power hence used for
surface sterilization in food diary hospitals to sterilize contaminated surfaces. UV rays produces Thymine
dimers in DNA which interrupt DNA replication.

II Ionizing Radiation: Shorter wavelength with high penetrating power. High energy radiations ionize
target molecules by removing valence electrons to form free radicals which causes nonspecific damage.

Gamma rays: 10-3-10-1nm.

High energy radiations emitted from radioactive isotopes such as Co 60. Due to shorter wavelength have
high penetrating power into biological tissues and are lethal to DNA by producing hyperactive free
radicals like superoxide, hydroxyl ion. δ rays used for commercial sterilization of packaged food and
medical devices. X rays and Cathode rays also employed.
Stains and Staining Techniques
Microbes are not visible to naked eye due to their minute size, transparent nature hence visibility is poor.
Staining increases the visibility of the microbes and provides clear contrast between microbes and
background.
Stains/ Dyes are chemical substances used to colour microbes. Stains may be Natural or Synthetic dyes
chemically organic compound containing benzene ring with a chromophore group.
Stains may be
Acidic Stains- They posesses –vely charged groups hence bind to +vely charged components.
Eg: Eosin, Acid fuchsin, Nigrosine.
Basic stains: They pocesses +vely charged groups which binds to –vely charged components such as
nucleic acid, proteins.
E.g: crystal violet, Safranin, methylene blue, malachite green.
Preparation of Smear
Process by which microbial cells are firmly attached in same condition and position to the surface of
glass slide.
Heat Fixation: Here microbial cells are fixed to the glass slide by gently passing over a flame.

Types of Staining:
■ Simple stains
■ Differential stains
■ Structural stains

1.Simple Staining: Direct/Positive Staining

It directly stains the surface of microbial cell.
Single basic stain is used. Cells appear colored under white background.
Negative Staining: It stains the background not the cell. Single acidic stain like Nigrosine, India ink are
employed. Microbial cell appear bright under dark background.
2. Differential Staining:
Differential staining differentiates between 2 types of bacteria based on staining characteristics. Different
bacteria react differently to a given stain based on their chemical nature. Hence it is defined as a method
of chemically distinguishing between the different types of bacteria.
Types:
■ 1.Gram Staining
■ 2.Acidfast Staining

1.Gram Staining:
Gram staining to classify all bacteria into Gram positive and Gram negative bacteria.
It was first developed by microbiologist Hans Christian Gram in 1884. The differential response of
bacteria to gram’s stain is related to chemical composition of cell wall of bacteria.
The Gram –ve bacteria has thin and complex cell wall with more lipid content and less peptidoglycon,
hence alcohol treatment dissolves the lipid results in formation of large pores through which basic stain
leaks out later cell takes counter stain and appear red.
The Gram +ve bacteria has thick peptidoglycon layer and less lipid content hence alcohol treatment
doesn’t allow the leakage of basic stain and appear violet.
■ Reagents
■ Basic stain/Primary stain- Crystal violet
■ Mordant- Gram’s Iodine
■ De colorizing agent- 95% Ethanol
■ Counter stain/Secondary stain- Safranin
■ Observation:
■ Bacteria appears violet are Gram + ve, bacteria appearing pink are Gram - ve.
2.Acid fast Staining:
It is a type of differential staining primarily for identification of Mycoplasma, tuberculosis bacilli &
leprosy bacilli and some Actinomycetes.
It was developed by Paul Ehrlich in 1882 to differentiate between Acidfast and Nonacidfast microbes.
This method modified by Ziehl-Neelsen.
It measures acid fastness of the bacteria i.e resistance of microbe to decolorizing acidic agents. Bacteria
which retains the primary stain after treating with strong acid and appears red called Acidfast and if
decolorised after acid wash, takes counter stain and appears blue called Nonacidfast. The property of
acid fastness is correlated with high Mycolic acid content in cell wall which requires harsh treatment.
■ Reagents
■ Primary stain- Carbol fuchsin
■ Decolorising agent- Sulphuric acid
■ Counter stain- Methylene blue.
 1. Structural Staining:
Flagella staining
Capsule staining
Endospore staining

Flagella staining:
Flagella are thin filamentous structure originate in the cytoplasm which helps in Locomotion of
bacteria. As flagella are thin structures, in order to observe them under microscope, they have
to be thickened by adding Mordant and later stained with a suitable stain.
Protocol: A smear of Proteus species is made on a glass slide and allowed to air dry.
Add mixture of Tannic acid (mordant) and Rosaniline dye, leave it for 10 minutes, wash, air dry.
Observe under microscope. Flagella stain Red.

Capsule Staining:
Many bacterial cells are surrounded by a mucilaginous substance forming a slimy coat around
the cells, this structure is known as Capsule.
It is known as slime layer when it is irregular or loosely bound.
Capsule is composed of polysaccharides and glyco proteins. This capsule offer virulence which is
important in pathogenesis.
Capsule is seen in some bacteria such as Streptococcus pneumoniae, Clostridium perfringens.
Smear of S.pneumoniae is made on glass slide, air dry.
Protocol: Apply Congo red solution on smear, allow to air dry.
Fix the smear with acid alcohol for 15 minutes, wash.
Stain with Acid fuschin for 1 min, wash.
Blot dry, observe under microscope.
Capsule appears colorless surrounding red bacterial cells against dark blue background.
Endospore Staining:
Some bacteria such as species of Bacillus, Clostridium form thick walled Endospore during un favorable
conditions. As it is difficult to stain endospores, special stains are required.
Protocol: Prepare smear of Bacillus subtilis, heat fix.
Cover the smear with 5% aq Malachite green, slide is heated to steaming for 5 minutes, wash.
Stain with counter stain 0.25% Safranin for 30 sec, wash, air dry and observe under microscope.
Endospore stain green & vegetative cells stain red.

MICROBIAL TAXONOMY
Microbial taxonomy is a branch of biology which deals with the classification,
nomenclature and identification of microorganisms according to their natural
relationship.
Classification is systematic arrangement of organisms into groups(taxa) & further
arrangement.
Nomenclature is assigning systematic scientific names to taxa.
Identification is placing an unknown organism in one of the established named
taxa.
Corolus Linnaeus introduced Binomial nomenclature where each organism is
named by two terms Genus and Species name.
E.g Bacillus subtilis
Species: distinct organism with certain characteristics or group of similar strains.
Strain: cell population arising from a single organism as a pure culture.
Genus: collection of different species.

Five Kingdom Classification:

R H Whittaker proposed 5 kingdom classification.
■ Kingdom Monera/Prokaryota-Bacteria & cyanobacteria.
■ Kingdom Protista-Protozoa & Algae.
■ Kingdom Fungi-Yeasts & molds.
■ Kingdom Plantae- Gymnosperms, Pteridophytes, Angiosperms.
■ Kingdom Animalia-Reptiles, Birds, Mammals.

Bacteria:
Prokaryotic cells, cell wall made up of peptidoglycon. It includes gram +ve, gram
-ve bacteria, mycoplasma, cyanobacteria & filamentous actinomycetes.
Bergeys Manual
In 1923 David Bergey at university of Pennsylvania published a manual for the
identification of bacteria called ‘Bergeys manual of determinative Bacteriology’
based on similarities in characteristics of bacteria.
Kingdom Prokaryota- Division I Cyanobacteria
Division II Eubacteria
Eubacteria contains 19 sections.
1.Phototropic bacteria
2.Gliding bacteria
3.Sheathed bacteria
4.Budding bacteria
5.Spirichetes
6.Spiral & curved bacteria
7.gram –ve aerobic rods & cocci
8.Gram –ve facultative anaerobic rods.
9.Gram –ve anaerobic cocci
10.Gram –ve cocci & coccibacilli.
11.Gram +ve anaerobic cocci
12.Gram –ve chemolithotrophic bacteria
13. Gram +ve cocci
14.Endospore forming rods & cocci
15.Gram +ve asporogenus rods
16.Actinomycetes & related organisms
17.Rikcettsiae
18.Mycoplasmas
19.Archae Ba, methanogenic, halophiles, thermophiles.

In 1984 Bergey’s manual updated & published in 4 volumes where prokaryotes

classified into 4 divisions.
■ Division I Gracilicutes: gram -ve
■ Division II Firmicutes: gram+ve
■ Division III Temricutes: without cellwall
■ Division IV Mendosicutes: without peptidoglycon

Nomenclature of Bacteria
Each species is designated by Latin binomial name, Genus name should start with
capital letter, Species name should be small letter. Both names should be
underlined if hand written and should be printed in italics. E.g: Escherichia coli
Author citation should be after name, B thuringiensis.Berciner
Bacterial Classification
I Based on Morphology:
A) Shape: 4 classes
1.Coccus- small Spherical cells occurs in clusters.
Monococci- occur solitary E.g: Micrococcus
Diplococci-occur in pair E.g: Diplococcus pneumoniae
Streptococci- attached in the form of chain E.g: Streptococcus pyrogens
Tetracocci-occur in 4. E.g: Tetracoccus
Staphylococcus-occurs in bunches E.g: Staphylococcus aureus
Sarcianae- cuboidal form. E.g: Sarcina
2.Bacilli: rod shaped bacteria. E.g: Ecoli, Bacillus subtilis
May be Diplobacilli, Streptobacilli
3.Spirillum: spiral shaped having turns showing helical structure. E.g:
Rhodospirillum
4.Vibrio: curved rods assuming comma shape.E.g: Vibrio cholerae

B.Flagella: It is filamentous structure helps in locomotion.

Depending on number and position of flagella
Atrichous- without flagella.
E.g: cocci
Monotrichous- with single flagella.
E.g :Pseudomonas aerugenosa
Lophotrichous- tuft of flagella at one end of the poles.
E.g: Pseudomonas fluorescens
Amphitrichous- tuft of flagella at both the ends.
E.g: Spirillum
Peritrichous- flagella present all over the surface.
E.g: Salmonella typhi

II Based on Nutrition:
1.Autotrophs: bacteria capable of synthesizing their own carbohydrates in presence of
sunlight(Photoautotrophs).
E.g: Chlorobium
2.Heterotrophs: cant synthesize food
2 subtypes:
a) Chemoorganotrophs: derive energy from organic carbon sources such as glucose, starch, lactose.
E.g: Ecoli, Bacillus, Salmonella
b) Chemolithotrophs: derive energy from inorganic sources such as ammonia, nitrate, sulphates.
E.g: Nitrobacter, Nitrosomonas, Methanobacillus.

III Based on Staining Reaction:

Based on Gram staining bacteria are classified into
Gram +ve Bacteria: Have thick complex peptidoglycon content in cell wall and less lipid, hence retain
basic stain crystal violet appear dark purple.
E.g: Bacillus, Staphylococcus, Lactobacillus
Gram –ve Bacteria: Have thin less peptidoglycon and more lipid content hence crystal violet leaks out
takes up counter stain Safranin appears red/pink.
E.g: Ecoli, Pseudomonas.

IV Based on Extreme environment:

Bacteria adopted for extreme environment such as hot springs, salt lakes, acidic, alkaline situations are
called Extremophiles.

A) Based on Temperature sensitivity:

Thermophiles: grows at high temperature above 60oc even upto 80-100oc.
E.g: Thermus aquaticus
Mesophiles: grows best at normal temperature range of 20-45oc.
E.g: Ecoli
Psychrophiles: grows at low temperature below 4oc.
E.g: Pseudomonas.

B) Extreme pH tolerance:
1. Acidophiles: Bacteria grows at acidic pH 0-4 less than 5.
E.g: Thiobacillus thioxidans
2. Alkalophiles: Bacteria grows at alkaline pH 8-11.5
E.g: Clostridium species
3. Halophiles: Bacteria capable of surviving at high salt concentration.
E.g: Halobacter

Pathogenic Microorganisms
Bacterial Diseases of Man
Airborne Bacterial Diseases: Tuberculosis

Causative organism: Mycobacterium tuberculosis

Mycobacterium are Acid-fast rods Grow slowly; some species are difficult to
culture
Transmission: Airborne Contact
• Prolonged Exposure
• Occasionally via skin contact or wounds

Symptoms: Lung Infection

• Destruction of alveoli
• Chronic Cough; sputum
• Tubercle Formation
• May remain dormant for years
and then become active again
• May spread to other areas of the body

Detection: Microscopic examination

• Chest X-Ray
• Culture
• Tuberculin Skin Test
•
Treatment: DOT therapy

Prevention: TB Vaccination BCG vaccine ( Bacillus Calmetto Guerin)

Water borne Bacterial Diseases:

1. Typhoid Fever
Causative organism: Salmonella typhi
a. Properties of the genus Salmonella
i. A member of the family Enterobacteriaceae
ii. Colon flora; sometimes carried asymptomatically
iii. Gram negative rods
iv. Facultatively anaerobic
Salmonella typhi -Most virulent member of the genus
Transmission via oral route
Water contaminated with sewage
often associated with contact with infected persons, either carriers

Symptoms:
Invades intestinal epithelium tissue
• ulceration
• bloody stools but little diarrhea
• Fever; delirium
• blood vessel hemorrhaging
• rose-colored spots on the abdomen
• bowel perforation
• gall-bladder infection
Treatment:
Antibiotic therapy
Chloramphenicol
Disease can be controlled by sewage treatment, water purification.

2.Water borne Disease: Cholera

Causative organism: Vibrio cholera

• Gram-negative curved bacteria
• comma-shaped, facultative anaerobic bacteria.
Transmission:
• Contaminated water and food
Symptoms : Gastroenteritis with extensive severe diarrhea
• Cholera enterotoxin
• Toxin blocks water reabsorption by inhibiting the anion active
transport mechanism(Na K pump) in large intestinal epithelium
• “Rice water” stools
• Dehydration & death
Treatment: Administration of body fluids, electrolytes through Oral Rehydration
Solution (ORS).
Antibiotic therapy
Good sanitation.

Soil born Bacterial Disease: Tetanus

Causative organism: Clostridium tetani
Gram + endospore (terminal) forming rod shared Soil bacteria
It is anaerobic, peritrichous grows at 37c and pH 7.4.

Transmission :
– Spores in soil enter through Wounds;
– Improperly sterilized surgical instruments.
–
Symptoms: Starts with the release of Tetanospasmin A neurotoxin
• Prevents release of inhibitory neurotransmitter Glycine leads to
the continuous nerve stimulation.
• Neurotoxin causes muscles to contract leads to painful muscle
spasms.
• Body stiffens and bend like an arc.
• Jaw muscles contracts causing Lock-jaw.
• Break bones, rigid paralysis and prevent breathing.

Treatment:
Before clinical symptoms treated with tetanus Antitoxin plus muscle relaxants.
• After toxin released , symptoms diagnosed it is too late to treat.

Prevention: Vaccination with tetanus toxoid as DPT triple Ag for Diphtheria,

Pertussis (Whooping cough) and Tetanus.
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