UNIT- II

(CHAPTER-6)
STAININGTECHNIQUES &
BÊOCHEMICAL TESTS

Points to be covered in this topic

1. INTRODUCTION

2. TYPES OF STAINING TECHNIQUES

3.GRAMS STAINING
4. ACID FAST STAINING

5. BIOCHEMICAL TESTS

NTRODUCTION
&STAINING TECHNIQUES
Staining is a technique used to enhance contrast in samples, generally at
the microscopic level.
"According to microbiological definitions, stains are dyes or reagents
used for differential colouring of clarity under microscope.
"Chemically a stain / dye is defined as an organic compound containing a
benzene ring plus a chromophore group.
 Types ofstains:
1. Acidic dyes: Anionic, stain the cytoplasmic components of cells which are
more alkaline in nature e.g, picric acid, acid fuchsin, eosin.
2. Basic dyes: Cationic and combine with those cellular elements which
are acidic in nature (nucdeic acids) e.g, methylene blue,crystal violet,
safranin.

3. Neutral stains: Formed by mixing together aqueous solution of certain

acidic and basic dyes.

TYPES OF STAINING TECHNIQUES

(Staining techniques

Simple staining (Different ial staining

Morphological shapes
and arrangements is Identification Visualization of structure
visual | "Spore staining
Gram staining Acis Fast staining Wall staining
"Capsule staining
Positive staining Negative staining "Flagella staining

* SIMPLE STAINING
Types of simple staining
r Positive staining: In positive staining, the stain (methylene blue) is basic
(cationic) having positive charge and attaches to the surface of the object
that is negatively charged.
Negative staining: n negative staining, the stain (India ink, nigrosine) is
acidic (anionic) having negative charge and is repelled by the object
(bacteria) that is negatively charged and therefore bacterial cell appears
transparent. The stain stains only background but not bacteria.
Simple staining involves single dye or staining reagent.
PRINCIPLE The purpose of staining is to demonstrate cell size,
shape and arrangement of bacterial cells.
 Since bacterial cells. usually have a negative charge on their surface, they
are most readily coloured by basic stains.
PROCEDURE- Reagent: Loeffler's methylene blue solution, iodine.
o Prepare smear of a given culture (add few amount of bacteria in one to
two drops of distilled water and mix it well) or material by spreading a
thin film on a clean glass slide.

o Dry it and then heat fix by passing the slide 2 to 3 times through the
flame with the smeared side facing upwards.
Step 1. Mede
Meat Fix
o Cover the smear with methylene Smear
Sod

blue and allow the dye to remain in

the smear for approximately one
Step 2.
minute. Staln

Saturate the smear again but this time with lodine. lodine will set the
stain.

Wash any excess iodine with gently running tap water.

o Apply oil directly to the smear, and focus the smear first under low
power objective and then under oil immersion objective
RESULT
The bacteria will appear blue or red depending
upon the stain used with characteristic
morphology and may be cocci, cacilli,
coccobacilli, spiral, comma shaped etc.

DIFFERENTIAL STAINING
Differential staining is a staining process which uses more than one
chemical stain. Using multiple stains can better differentiate between
different microorganisms or structures/cellular components of a
single organism
 Tynes of differentialstaining staining
Grams staining
Acid fast staining
1. GRAM STAINING
Gram staining is one of the most widely used technique
for the differentiation and identification of bacteria.
This differential staining technique was discovered by
Dr. Christian Gram in 1884.
It not only reveals the size and shape of bacteria but is also used to
differentiate bacteria into Gram-positive and Gram-negative cells.
Hence, it is known as differential staining
PRINCIPLE
Gram staining differentiates bacteria by the chemical and physical
properties of their cell walls by detecting peptidogtycan, which is
present in the cell wall of Gram-positive bacteria.
Gram-negative cells contain a very small layer of peptidoglycan that is
dissolved when the alcohol isadded. This is why the cell loses its initial
colour from the primary stain.
Gram-positive bacteria retain the crystal violet dye, and thus are stained
violet, while the Gram-negative bacteria do not and it gives pink colour
after adding counter stain
Reagents used:
Primary stain- Crystal violet
Mordant- Grams iodine, (A mordant nmay be defined as 'any substance
that forms an insoluble compound with stain and serves to fix the
colour to bacterial cell.
It leads to form crystal violent iodine magnesium ribonuclease (CV-1
Mg ribonuclease] complex in Gram-positive bacteria.
This complex is not formed in Gram-negative bacteria, as Mg-ribonucleate
is absent in the cell wall. Hence, only CV-1 complex is formed in Gram
negative bacteria.)
 Decolourising agent- Ethyl alcohol, On application of decolourisingagent
like alcohol or acetone, shrinkage of cell wall takes place due to
dehydration and decreases the permeability for CV-1-Mg ribonucleate
complex.
Thus, the complex is retained in the cell and hence cell is stained deep
violet in colour.
On the other hand, the treatment of decolourising agent extracts lipid from
cell wall of Gram-negative cell and there is increase in permeability
property of cell wall. Due to this, CV-1 complex is extracted and cells
gets decolourised (lose violet colour).)
Counter stain- Safranin, (This is the final reagent to stain red, those cells
that have been previously decolourised. Since only Gram-negative cells are
decolourised, they may now absorb the counter stain. Gram-positive cells
retain the violet colour of the primary stain. )
PROCEDURE oUTER E
Make smears of a given
PEPTOOOUCAN
culture on a clear glass slide. PEPLAMC SAACE

o Air dry the smear and heat fix it. GRAM POSITIVE GRAM NEGATIVE

o Cover the smear completely with crystal violet stain and leave the stain
on the slide for one minute.
Gram Posltve Gram log

Focaton
o Wash the slide gently in distilled
water or tap water.
Cystal iwolt
o Flood the smear with Gram (or lodine trootn¡nt
Lugol's) iodine solution and wait for
one minute. Wash with tap water Decolirzibon
gently and drain carefully.
Counler soin
seranin

o Add ethyl alcohol (9596) or alcohol-acetone (1:1) solution drop by drop.

until the smear becomes free from any colorization
o Wash the slide gently under running tap water and drain.

Now counter stain with safranin and wait for 30 seconds.

o Wash again and blot dry with blotting paper or simply air dry the slide
and observe under oil immersion objective.
EXAMPLES
Gram negative bacteria: Xanthomonas, acetobacteracea(vinegar),
Escherichia coli, Klebsiella pneumonia, salmonella, pseudomnonas
aeruginosa.
Gram positive bacteria: lactobacillus acidophilus, Bifidobacterium,
staphylococcus aureus, anthrax, mycobacterium tuberculosis
RESULT
Bacteria that appear blue/violet /purple are assigned as Gram positive
while as those appearing red/pink are assigned as gram negative.

2. ACID FAST STAINING

The technique was developed by Paul Ehrlich(1882) and was modified
later by Zichl Neelsen and therefore also known as Zichl-Neelsen
staining.
This is a differential staining used to identify mainly the
members of Mycobacteriumn especially Mycobacterium
tuberculosis and Mycobacterium leprae.
These organisms are difficult to satin by ordinary
staining methods due to presence of high lipid content
(mycolic acid) in their cell wall.
BACTERIAARE CLASSIEIED AS:
(M Acid fast if they retain the primary stain (carbol fuchsin) after the
application of strong acid and appear red.
(il Non-acid fast:_If they do not retain the primary stain and are
counterstained by methylene blue.
 PRINCIPLE
When the smear is stained with carbol fuchsin, it solubilizes the lipoidal
material present in the Mycobacterial cell wall but by the application of
heat, carbol fuchsin further penetrates through lipoidal wall and enters
into cytoplasm. After that all cell appears red.
Then the smear is decolorized with decolorizing agent (3% HCL in 95%
alcohol) but the acid fast cells are resistant due to the presence of large
amount of lipoidal material in their cell wall which prevents the
penetration of decolorizing solution.
The non-acid fast organism lack the lipoidal material in their cell wall
due to which they are easily decolorized, leaving the cells colourless.
Then the smear is stained with counterstain, methylene blue.
Only decolorized cells absorb the counter stain and take its colour and
appears blue while acid-fast cells retain the red colour.
PROCEDURE
o Prepare a smear of a purulent portion
of the specimen on a clean glass slide.
o Air dry and heat fix the smear. AID FAST NON-AGID FAST
BACTERIA RED BACTERLA BLUE

o Flood the smear with freshly filtered carbol fuchsin till steam rises.
Continue to heat for 5 minutes so that steam is seen but without boiling. Do
not allow the side to dry and add more stain from time to time to prevent
this drying
o Cool and wash the stain off the slide with water.
o Cover the side with acid alcohol solution for 3
minutes. Wash with tap water and drain.
Repeat decolourization process until smear
becomes faint pink. Finally wash with water.

o Cover the slide with methylene blue stain and leave it for 2 minutes. Wash
with tap water, blot d1y or air dry the slide and observe under oil
immersion objective.
 RESULT
Acid fast organisms will appear bright red on a blue background while
as non-acid fast organism will appear dark blue in colour.

SPORE STAINING
Spore staining technique is used for detection of spore and type of
spores. It isnot easy to stain spore by primary stain malachite greens.
The smear is heated to accept the stain. Once the spore accepts the
malachite green, it cannot be decolourised by tap water
Vegetative cells easily get decolourized by tap water
and take counter stain and appear red (safranin).
This method is called Schaeffer and Fulton method
modified by Ashby.
Donor method is also used for spore staining in which carbol fuchsin is
used as primary stain. After heating the side with stain for 5 to 10
minutes, wash it and perform negative staining.
CAPSULE STAINING
Capsule staining is used to detect capsules. It can be performed by using
positive as well as negative staining techniques.
" Due to the non ionic nature of capsule, it has very less affinity for dye.
" In positive stains, crystal violet is applied which
stains bacterial cell and also copper sulphate is
applied and it allows the stain (light violet) to retain.
Capsules are also easily demonstrated by negative staining techniques.
When stain solution is applied, it stains the cell and on application of
nigrosine background is stained leaving the capsule colourless.
FLAGELLA STAINING
Flagella staining is used to detect presence and
arrangement of flagella and cytoplasmic inclusion.
Staining is used to identify intracellular deposits of starch, glycogen,
polyphosphates, Hydroxyl butyrate and other substances.
 OBIOCHEMICAL TESTS (IMVIC)
The IMVIC series is a group of four individual tests that are commonly
used to identify bacterial species, especially coliforms.
Acoliform is a gram negative, aerobic anaerobic rod which produces gas
from lactose within 48 hours. The presence of some coliforms indicate
faecal contamination.
Each of the letters in "IMViC" stands for one of these tests.
"I" is for indole; (test for production of indole from tryptophan)
"M" is for methyl red;"(methyl red test for acid production from glucose)
"y" is for Voges - Proskauer, (test for production of acetoin from
glucose)
"C" is for citrate,(test for use of citrate for sole carbon)
Lowercase" (i) is added for the ease of pronunciation.
IMVIC is an acronym that stands for four different tests
PURPOSE FOR TEST
To find out the procedure of treatment or prevention of a disease we
need to find out the causative bacteria responsible, this methods helps to
detect the bacteria.

All the bacteria present in intestine of a human or other mammals belong

to the family of Enterobacteriaceae. These enterobacteria are short,
gram-negative, and non sporing bacilli.
The identification of these fermentative and non fermentative bacteria can
be accomplished by IMViC test.
In medical research, it will help to identify the pathogen responsible for
the disease.

1. INDOLE TEST
PRINCIPLE
Some bacteria can produce indole from amino acid tryptophan using
the enzyme typtophanase.
 Typtophanase
Trptophan water Indole + Pyruvic acid +NH,
Production of indole is detected using Ehrlich's reagent or Kovac's
reagent.
Indole reacts with the aldehyde in the reagent to give a red colour. An
alcoholic layer concentrates the red colour as a ring at the top.
º PROCEDURE
Bacterium to be tested is inoculated in peptone water, which contains
amino acid tryptophan and incubated overnight at 370C. Prepare 1%
tryptophan broth are one.
o Incubate one set of test tube with test organism and maintain one set as
negative control without inoculation . inoculate one set of test tube with
E.coli use as positive control.
o Following incubation few drops of Kovac's reagent are added. Shake
gently.
o Kovac's reagent consists of para-dimethyl amino benzaldehyde 10 gm,
isoamylalcohol 150gm and con. HCI 50 ml.
o Allow the tubes to stand for 2 min. so that the reagent comes to the top and
then compare test culture with the control tubes.
o Ehrlich's reagent is more sensitive in detecting indole production in
anaerobes and non-fermenters.

RESUT
Formation of a red or pink coloured ring at the top is
taken as positive.
o Escherichía coli: Positive;
o Klebsiella pneumoniae: Negative
 2.METHYL RED TEST
Bacteria which produces add through fermentation of glucose can be
identifîed using this test. Methyl red indicator produces red coloration in
acidic pH while it produces orange coloration in non-acidic PH.
PRINCIPLE
The basic principle of MR test is to check the ability of the organism to
produce andmaintain sufficient amount of stable acid as end product
from glucose fermentation and to overcome the buffering capacity of the
system.

Among different fermentation pathway followed by bacteria, MR test works

upon the mixed acid fermentation pathway.
Glucose-> 2 pyruvate---> Succinic acid, lactic acid, acetic acid,
formic acid + CO2 + H,0

These acids lowers the pH of the medium which is nearly neutral so that
the methyl red indicator changes its colour to red.
The pH range of the methyl red indicator is 4.4-6
but if there is no or low production of acids, the pH of the medium remains
unchanged and the indicator shows yellow colour.
Hence, the red colour of the broth medium after adding indicator is
positive MR test.
PROCEDURE
o Prepare MRVP broth in test tubes. Inoculate the broth aseptically with 2
loop full of respective bacterial culture.
o Label the test tubes with name of organism inoculated.
o Incubate the test tubesat 37°C for 48-72 hours.
o Add few drops of methyl red indicator
in the incubated tubes.

RESULT MR test positive: bright red colour (E.coli)

MR test negative: yellow colour (Klebsiella )
3. VOGUES PRUSKAUER TEST
The Voges-Proskauer (VP) test is used to determine if an organism
produces acetyl methyl carbinol from glucose fermentation.
PRINCIPLE
The VogessProskauer (VP) test is used to determine if an onganism
produces acetylmethyl carbinol from glucose fermentation. If
present, acetylmethyl carbinol is converted to diacetyl in the presence of
o¢- naphthol, strong alkali (40% KOH), and atmospheric oxygen.
PROCEDURE
o Prior to inoculation, allow medium to equilibrate to room temperature.
o Using organisms taken from an 18-24 hour pure culture, lightly
inoculate the medium.
o Incubate aerobically at 37 degrees C. for 24 hours.
Following 24 hours of incubation, aliquot 2 ml of the broth toa clean test
tube.
o Re-incubate the remaining broth for an additional 24 hours.
o Add 6 drops of 5% alpha-naphthol,and mix well to aerate.
o Add 2 drops of 40% potassium hydroxide, and mix well to aerate.
Observe for a pink-red colour at the surface within 30 min. Shake the
tube vigorously during the 30-min period.
RESULT
Positive Reaction: A pink-red colour at the surface
Examples: Viridans group streptococci (except Streptococcus
vestibularis), Listeria, Enterobacter, Klebsiella, Serratia marcescens, Hafnia
alvei, Vibrio eltor, Vibrio alginolyticus, etc.
Negative Reaction: A lack of pink-red colour
Examples: Streptococcus mitis, Citrobacter sp, Shigella, Yersinia,
Edwardsiella, Salmonella, Vibrio furnissii, Vibrio fluvialis, Vibrio vuinificus,
and Vibrio parahaemolyticus etc.
Acopper colour should be considered negative.
Arust colour is a weak positive reaction.
 4. CITRATE UTILIZATION TEST
Citrate testing is used to determine the ability of the bacteria to use
sodium citrate as the only source of carbon and inorganic ammonium
hydrogen phosphate (NH,H, PO,) as a source of nitrogen.
PRINCIPLE
This test detects the ability of an organism to utilize citrate as the sole
Source of carbon and energy.
Citrate Oxaloacetate + acetate
Oxaloacetate Pyruvate + CO,
CO, + Na + H,0 ’ Na,C03

Production of Na2CO3 as well as NH3 from utilization of sodium itrate and

ammonium salt respectively results in alkaline ph.
This results in change of medium's colour from green to blue.
PROCEDURE
o Consider a slant, inject Simmons citrate agar genty on the slant by
slightly touching the tip of the needle to the colony, which is 18 -24 hours
old.
o In the citrate medium, organisms require more time to grow. Therefore,
the solution must be incubated at 35°C to 37°C for 18- 24 hours.
Some organisms may also require seven days of incubation.
o Observe the colour change in the solution, the development of blue colour
indicates the alkylation process.
RESUT
Positive result:
The reagent used - Bromothymol blue, Growth on the medium indicates
the change in the colour of the reagent from green to blue.
Negative result:
Very less or no growth in the medium and
colour of the solution remains the same.
 UNIT- II
(CHAPTER-7)
STERILIZATION TECHNIQUES

Points to be covered in this topic

1. INTRODUCTION

2. TYPES OF STERLIZATION METHODs

3. PHYSICAL STERLIZATION

4. RADIATION STERLIZATION

5. CHEMICAL STERLIZATION

6.MECHANICAL STERLIZATION

DINTRODUCTION
STERLIZATION
" It is the process by which an article, surface or medium is made free of all
microorganisms either in vegetative or spore form.
" It is the process of absolute kill of microorganisms.
 NEED FOR STERLIZATION
The growth of microorganisms can be controlled by two basic ways: -
By either killing the microorganism,
Or by inhibiting the growth of microorganism.
This is achieved by using physical or chemical agents which either kill or
prevent the growth.
The agents which kill the microorganisms are called 'cidal' agent
&the agents which inhibit the growth (without killing them) are called
static agents.
Bactericidal-agents killing bacteria
V Virucidal-agents killing viruses
otore
/ Fungicidal-agents kiling fungi
TYPES OF STERLIZATION METHODS
Sterilization

(Physical) Chemical Mechanical

Gas
Formaldehyde Filteration
Sunlight Hat Vibration Radiation - Ethylene oxide - Asbestos
lonization -B-proplolactone - Sintered glass
Dry Heat Moist heat -X-ray Ozone -Eaethen ware
-Inceneratdon Temperature Gamma rays +Liquid - Candle
-Hot alr oven below 100°c Alcohol - Membrane
-Flaming " Steam Nonlonization Aldehydes - Air filter
-Red Heat - IR ionization Phenollcs -Syringe filter
" Pasteurlzation
-UV rays Halogens
" Inspissation Heavy Metals
-Temperature -Surfactants
at 100°C -Dyes
Tyndalization
"Boling
Temperature
above 100°c
"Autoclave
 PHYSICALSTERLIZATION
These methods involve processes by the use of physical means. They may
involve the utilization of heat in the presence or in the absence of
moisture or the applications of radiations or mechanical fltration.
1,. DRY HEAT:
Dry heat kills microorganisms by causing protein denaturation &
destructive oxidation of essential cell constituents.

Microorganisms are more resistance to dry heat thus this process requires
higher temperature and longer exposure time.
> INCENERATION
Incineration is the process of sterilization along with a significant reduction in
the volume of the wastes.
The scraps are heated till they become ash which is then disposed of later.
This process is conducted in a device called incinerator
PRINCIPLE :_It kills microorganisms by destructive oxidation of
essential cell constituents.
ADVANTAGES:
Potentially destroy any material containing organic carbon including
pathogens.
Typically reduce the volumne and mnass of material that must be
disposed off in landfills by 80 to 95 percent.
Heat of combustion can be recovered and used to
generate steam or hot water.
DISADVANTAGES:
Incineration may emit traces amounts of unwanted pollutants such as
the polychlorinated dibenzo dioxins and furans and matter particulates
if incinerators are not well designated and operated.
The ash and waste water produced by the process also contain toxin
compounds, which have t be treated to avoid adverse effects on health
 and the environment.

EXAMPLES: This is an excellent method for rapidly

destroying materials e.g. pathological materiel,
bedding, animals carcassec soiled dressing etc
Polystyrene type of materials emit
clouds of dense black smoke and hence should be autoclaved in appropriate
containers.
FLAMING
Flaming is a type of dry sterilization that involves exposure of metallic
objects to flame for some time where the flame burns microbes and other dust
presents in the instrument.
In the case of flaming, the instrument is dipped in alcohol or spirit before
burning it in a gas flame.
PRINCIPLE:It kills microorganisms by destructive oxidation of essential
cell constituents. ie: Glass slides, scalpels,and mouths of culture tubes
or conical flasks are passed through Bunsen flame without allowing them
to become red hot.

RED HEAT
Red heat sterilization is the process of instant sterilization by
holding the instruments in a Bunsen flame till they become
red hot.
It isused to sterilize metallic objects by holding them on a flame till they are
red hot. EX: inoculating wires, needles, forceps etc.
HOT AIR OVEN
PRINCIPLE: Jt kills microorganisms by destructive oxidation of
essential cellconstituents.
CONSTRUCTION:
The modern hot air ovens consist of a double walled chamber of
aluminium or stainless steel separated from the outer case by a thick
layer of insulation made of fiberglass.
Insulation is also filled in the hollow
flanged door, which carries an ed
asbestos jacket that provides a Troy or

tight seal.
Heating is affected by electrical
heating elements and thermostats
automatically control temp.
WORKING:
The sterilization temperature &holding time for sterilization in hot air
oven is 160°C for two hours is required for sterilization. 180°c for 30
minutes.
"3 hours at 140°C. "1 hour at 160°C. "30 minutes at 1800C.
ADVANTAGES:
Adry heat cabinet is easy to install and has relatively low operating costs.
It penetrates materials.
It Is nontoxic and does not harm the environment.
It is non-corossive for metal and sharp instruments.
Does not require water.
DISADVANTAGES:
Time consuming method because of slow rate of heat penetration and
microbial killing.
High temperatures are not suitable for materials like rubber, plastic, etc.

APPLICATION:
Used to sterilize the equipment's like glassware, forceps, scissors,
spatula, swabs, some pharmaceutical substances such as glycerin, fixed
oil, paraffin propylene.
Used in many industries for drying and baking and curing process.
Used tosterilize powders and other non volatile compounds.
2. MOIST HEAT:
r PRINCIPLE
Sterilization by moist heat means killing of microorganisms with hot
water or steam. The lethal effect of moist heat is due to the denaturation
and coagulation of proteins.
iº ADVANTAGES:
High heat content plus rapid heat transfer.
Destroys micro-organism more efficiently than dry heat.
It can be used for a large number of injections, Ophthalmic solutions,
irritants, dialysis luids etc.
It rapidly penetrates porous materials and is therefore very suitable for
sterilizing surgical dressings and materials.
The process is adaptable for plastic containers and some other special
dosage forms.
º DISADVANTAGES:
It is not suitable for anhydrous materials such as powders and oils.
It cannot be used for thermolabile substances.

It does not destroy pyrogen's.

Temnerature helow 100 degree Celsius:
PASTEURIZATION
Pasteurization is done with milk.
There are two different types of pasteurization methods that are used
for sterilization of milk; Holder method (63°C for 30 minutes) and
flash method (720C for 20 seconds).
This method is effective against all non-sporing pathogens such as
mycobacteria, Salmonella, etc. except Coxiella burnetii which
survives the holder method due to heat resistant characteristics.
 Similarty, serum and body fluids with congealable proteins are also
sterilizedat 56°C for 1 hour in water baths.

INSPISSATION
By this method slow solidification of serum or egg is carried out at 80°c
to 85°C for one hour in an inspissator e.g. Lowestein-Jensen's medium,
Serum slopes etc.
This heating is used to solidify & not sterilize it.
However, if the process is repeated for days, some
degree of sterilization is achieved & is called
fractional sterilization.

* Temperature at 100 degree Celsius:

TYNDALLIZATION
Tyndallisation is also called as intermittent sterilization or fractional
sterilization. Tyndallisation is a method that is used for sterilization of
media with sugar and gelatin at 100°C for 30 minutes on three
success0ve days so as to preserve sugar which might be decomposed at a
higher temperature.
It is not recommended for sterilization of instrumnents. This method is
used to sterilize egg or serum, gelatin etc.
º BOILING
Boiling for 10 to 30 minutes kills mostly the vegetative forms of
bacteria, fungi and viruses at 50-70 degree in short time, but many spores
withstand boiling for considerable time.
Therefore boiling is not adequate for sterilization purpose.
This is not suitable for absolute sterility. Glass syringe, tubes, rubber
stopper or surgical instruments are sterilized by this method.
* Temperature ahove 100 degree Celsius:
AUTOCLAVE
The autoclave works on the principle of moist heat sterilization. The high
pressure inside the chamber increases the boiling point of water for the
sterilization of equipment.
> PRINCIPLE
The higher pressure also ensures the rapid penetration of heat into the
deeper parts of equipment. The moisture present in the steam causes
coagulation of proteins of microbes causing irreversible loss of their
activity and functions.
º CONSTRUCTION
The laboratory autoclave or pressure cooker type autoclave consists of a
vertical or horizontal cylinder of gun metal or stainless steel in a
supporting frame or case.
The lid is fastened by screw clamps and
The
rendered airtight by a asbestos gasket. The
autoclave has on its lid or upper side a Proroted
discharge tap for air and steam a pressure Tbe

rd
gauge and a safety valve that can be set to Heag
blow off at any desired pressure.
WORKING
OThe steam circulates within the jacket & is supplied under high
pressure to closed inner chamber where articles for sterilization are kept.

O One fifth of the cylinder is filled with water, materials to be sterilized are
kept inside, lid closed & heater is put on. Safety valve is adjusted to
required pressure.

O The boiling of water inside the chamber after sometime results in steam
which is allowed to escape with air mixture fill the cylinder becomes air
free.

OThe discharge tap is closed and the desired pressure inside in chamber
is allowed to rise to the one chosen for autoclaving for a fixed time Is
thus complete sterilization achieved.
Standard operating procedures for autoclaving is: 115 °C, half an hour. 121°C,
15 minutes.
S.NO TIME TEMPERATURE PRESSURE
in degree
Celsius
80 min 110 O Ibs
2 60 min 110 5 lbs
3 40 min 116 10 lbs
4 15 min 121 15 lbs
5 10min 126 20lbs
6 3 Min 130 25-30 bs

ADVANTAGES
Rapid and effective
Effective for sterilizingcloth surgical packs and towel packs. t
Less timne consuming.
Applicable for both thermoplastic and thermolabile
components.
> DISADVANTAGES
Items sensitive to heat cannot be sterilized
It tends to corrode carbon steel bum and instruments.
It is costly technique.
APPLICATIONS
It is used to sterilize anything, which is not injured by steam and high
temperature of sterilization. These includes, Agqueous parenteral
solutions e.g. distilled water, saline solutions
Aqueous liquid media e.g. liquid media with or without carbohydrate
and gelatin.
 Surgical dressings and fabrics.
Plastic and rubber closures.
Metal instruments.

Glass apparatus and containers.

RADIATION STERLIZATION
Sterilization by radiation is also called as cold sterilization because
ionizing radiations produce relatively litle heat in the material being
irradiated.

It is possible to sterilize heat-sensitive substances.

Based on their wavelength and penetration power, radiation can be
divided into two categories as non-ionizing radiations& ionizing
radiations.

Non - ionizing radiations have less energy and do not disturb the
atomic configuration ofthe target molecule.
> lonizing radiations - have energy and ionizing the target molecules.
V PRINCIPLE
Breakage of DNA and degradation of enzymes lead to the
death of the irradiated cells.

/ ADVANTAGES
No degradation of media during sterilization, thus it can be used for
thermally labile media.
Leaves no chemical residue Administration of precise dosage and
uniform dosage distribution.
Immediate availability of the media after sterilization.
DISADVANTAGES
This method is a more costly alternative to heat
sterilization.

Requires highly specialized equipment.

1. Jonizing radiations
They have very high penetration power and considerable energy thus it
can be used to remove bacterial spores.
XRAYS
X"rays are lethal to microorganisms and higher forms of life but are rarely
used in sterilization because their production is expensive and efficient
utilization is difficult (since radiations are given off in all directions from
the point of origin). Radio Weves
Microwaves
Infrared
GAMMARAYS Vsible Light
Gamma radiations are high-energy Uhraviolet
X-Rays
radiations emitted from certain radioisotopes Gamma Rays
such as Caesium-137 (137Cs) and Cobalt-60 (°Co), both relatively
inexpensive bioproducts of nuclear fission. Gamma rays are attractive for
use in commercial sterilization of materials of considerable thickness or
volume, e.g. packaged food or medical devices.
ADVANTAGES OF IONIZING RADIATION
High penetrating power, Rapidity of action, Temperature is not raised
,Flexibility regarding size, density,matter of state.
V DISADVANTAGES OF IONIZING RADIATION
Capital costs are high and specialized facilities are often needed e.g. for
gamma irradiation.

Use of gamma radiation requires handling and disposal of radioactive

material

Not compatible with all materials and can cause breakdown of the
packaging material and/or product.
APPLICATIONS OF IONIZING RADIATION
Sterilization of medical products (e.g. insulin syringes)
Sterilization of blood products , pharmaceutical products.
 Preservation of foodstuffs (spices etc)
Irradiation of cell cultures for research purposes.
2. Non- ionizing radiations
X-ray: gamma rays and cathode rays are highly lethal to DNA and other
vital cell constituents. since non-ionizing rays are of low energy and
have poor penetration power.
They are exposed on the substance to remove bacteria and
microorganism

ULTRAVIOLET RAYS
OMany cellular materials induding nucleic-acids absorb
ultravioletlight.
O It causes bonding of two adjacent pyrimidines i.e., the
formation of pyrimidine dimer.
O Resulting in the inhibition of DNA replication. This leads to
mutation and death of exposed organisms.
Ultraviolet radiation is used for disinfecting enclosed areas such as
bacterial laboratory, nurseries, inoculation hood, laminar flow, and
operation theatres.
Conventional UV light can penetrate and damage skin and also cause
cataracts.

Does not penetrate paper, glass, and cloth.

INERA- RED RAYS

Infra-red rays are low energy type electromagnetic rays, having
wavelengths longer than those of visible light. They kill microorganisms by
oxidation of molecules as a result of heat generated.
Infra-red rays are used for the rapid mass sterilization of syringes and
catheters.
 CHEMICALSTERLIZATION
Chemical methods are easy and economic-friendly. They are of two
types: gaseous and liquid.

LIQUID
Liquid sterilization destroy the microbes permanently.
ALCOHOL
Usually, 70% of alcohols are used as a chemical to kill bacteria. Methyl
alcohol, isopropyl alcohol, and ethyl alcohol are some important
chemicals used in this method.

HALOGENS
Chlorination can impact the bacteria directly. The blend of iodine
compounds and chlorine compounds can act as an antiseptic. Chlorine
compounds are hydrochloride, chlorine bleach and iodine compounds are
tincture, iodine, and iodophors.
ALDEHYDE
About 40% fornaldehyde solution is used as surface disinfection.
Formaldehyde and Glutaraldehyde are some of the best aldehydes used
in this process. Similarly, 50% phenol can be used.
HEAVY METALS
Heavy metals such as copper sulfate, mercuric salts, silver nitrate,
mercuric chloride are used in the sterilization method. Similarly, dyes
like aminacrine, acriflavine, acridine dyes are used to interact with
bacterial nucleic acids.

2. GASEQUS
Gaseous sterilization may be defined as the destruction of all living
microorganisms withachemical in a gaseous or Vapour state.
Although ethylene oxide is the most widely used gaseous sterilization
agent. other chemicals used are formaldehyde and Bpropiolactone.
In addition to these, various glycols, methyl bromide and alcohol have
been used for room sterilization.

> FORMALDEHYDDE
This gas is generated by heating a concentrated solution of
formaldehyde.
Formaldehyde in aqueous solution isknown as formalin and contains 37
to 40% formaldehyde.
Vaporization of formaldehyde, either from formalin or paraformaldehyde,
is used to sterilize an enclosed area.

" It combines readily with vital organic nitrogen compounds such as

proteins and nucleic acids. It is a bactericidal agent with poor
penetrating power. It kills both vegetative cells and spores.

> B-PROPIOLACTONE
It is capable of killing all microorganisms and is very active against
viruses.
It is highly bactericidal and used in concentrations of 2 to 5 mg/litre.
> ETHYLENE OXIDE
Action of ethylene oxide is due to its power of alkylating the amino,
carboxyl, hydroxyl and sulfhydryl groups in the enzymes and protein
molecule. It reacts with DNA and RNA.

Ethylene oxide is a powerful sterilizing agent for heat and moisture

sensitive materials.

It can be used for sterilization of medical and biological preparations,

catgut, plastic equípment's, antibiotics, plaster bandages, culture media,
hospital bedding, food stuff, heavy equipment, books, clothing and soil.
MECHANICALSTERLIZATION
FILTRATION
1. This method is used for sterilization of liquid substances or fluids such as
sera &solutions of heat-labile substances such as sugars, urea, enzyme
 &antibiotics which get damaged by heat process.
2. The method is also used for separation of bacteriophages & bacterial
toxins from bacteria.

3. The following types of filters have been used for sterilization of different
pharmaceuticals.
MEMBRANE FITERS
The membrane filters are made of cellulose
gloding g
material.
The membrane should be placed between needle and StalnlessM
syringe while sterilizing the substance. Rubber s

This filter is effectively used in the sterilization of

gas, solvent, and fluids.
nasembled Assembled
SEITZ FILTERS
The Seitz filters are made of asbestos material
nelr botto
thus it has thick structure and strong enough to AMer
er fek
filter the solution. When solution passes through
the Seitz flter, filter pad absorbs it and leaving
the bacteria and residues on the top of the
filter.
SHINTERED GLASS FILTERS
Since the sintered glass flters are made of glass, it
doesn't absorb liquids during filtration. The
main drawback of practicing this method is that
the flteris very soft and brittle and tend to
breakeasily.
CANDLE FILTERS
This modern mechanical filter is made of diatomous mud. It has minute
pores that have the tendency to absorb microbes. When the fluid passes
through the filters, microbes get stuck in the pores of candle filters.
S.NO CHEMICAAENT MECHANISM OF ACTION APPLICATIoN
1 Phenol and Phenolics Plasma membrane; enzyme For instruments, mucous
denaturation and Inacttvation. membranes, skin sur-faces, and
environmental surfaces.
2 Chlorohexidine Plasma membrane disrupted.
3 Halogens 1. lodine inhibits protein Used to disinfect glass wares,
function, a strong oxidising utensils, water.
entity.
2. Chlorine formns the strong
oxldizing agent
hypochlorous acld that
changes cellular
constituents.
Alcohol Liptd dissolution and proteln Disinfecting Instruments.
denaturation.
5 Heavy metals and Denaturation of enzyme and Used as germicidal and
derivatives. other essential proteins. antiseptlcs.
6 Surface active agents 1. Acid anionic detergent Powerful antiseptics.
cnzyme disruption and
inactivation may take place.
2 Catlonic detergents- protein
denaturation, enzyme
inhibition, disruption of
plasma membrane.
Organic acids Metabolic Inhibitlon- largely Effective at low PH, effective
affecting moulds. against microbes.
Aldehydes Affords protein denaturation Sterilizing medical equipment's
 UNIT- II
(CHAPTER- 8)
STERLITY EVALUATI(

Points to be covered in this topic

1. INTRODUCTION

2. MEMBRANEFILTRATION
3. DIRECT INOCULATION FILTRATION

4. EQUIPMENTS EMPLOYED IN LARGE

SCALESTERLIZATION

5. STERLITY INDICATOR

OINTRODUCTION
Sterility test is defined as Microbiological test applied to sterile product
to show are products manufactured and processed under specification
guided by cGMP Or to confirm the products either sterile or
nonsterile.

Evaluation of efficiency of sterilization/Sterility testing Sterility tests can

be carried out using following methods:
 Method A: Membrane filtration.
Method B: Direct Inoculation.

Membrane filtration
O First clean the membrane filter unit and sterilize the unit and
membrane filter separately by moist heat sterilization.

O Transfer the unit on lamínar air flow bench or aseptic area and place
the membrane filter in the unit.

OPass all the solution through filter under vacuum._.

O After filtration, the membrane is removed aseptically and cut into two
parts using sterile scissors.

o One half part of membrane filter is placed in 100ml of fuid thioglycollate

medium (FTM) and incubate at 30-350C for not less than 7 days.

O Other half part of membrane filter is placed in soyabean casein digest

medium (SCDM) and incubate at 20-250C for not less than 7 days.
Observe the turbidity in the medium by comparing with the standard tube. If
it has no turbidity in fluid thioglycollate medium and soyabean casein
digest medium means it is free from microorganisms and suitable for use.
r Directinoculation filtration
Cotton or prefilled syringe is transferred directly to culture media using
sterile instruments such as sterile forceps.
O Incubate sample containing fluid thioglycollate medium (FTM) at 30
350C for not less than 7 days and soyabean casein digest medium
(SCDM) at 20-250C for not less than 7days.

O Observe the turbidity in the medium by comparing with the standard

tube.

O If it has no turbidity in fluid thioglycollate medium and soyabean casein

digest medium means it is free from microorganisms and suitable for
use.

Thermocouples:
An autoclave's temperature can be measured using thermocouples. A
particular autoclave temperature is deemed to be the appropriate
temperature for sterilization,
Brown Tubes:
Along with the articles, these tubes go into the autoclave. When the
temperature inside the autoclave reaches 121°, these tubes turn green,
which helps determine whether the articles are properly sterilized.
r Bacillus Stereo thermophilus Spores
These spores must be exposed to 121°C for 12 minutes in order tobe killed.
Within the envelope are placed paper strips containing 106 spores before
being placed in the autoclave. Inoculated with culture media, these strips are
autoclaved and then inoculated. It is determined if these spores are not
growing in the culture media it means sterilization has been properly
performed.
r Autoclave Tane
In the autoclave,the product changes color when heated to 121°C due to
lead carbonate. To check whether the sterilization process has the abílity
to kill bacteria (microorganisms), and then to ensure that all
microorganisms have been killed.
" This can be measured by the following three factors:
> Dvalue (decimal reduction timel:
The D value indicates the time in minutes it takes to destroy 90% of
viable microorganisms at a constant(defined) temperature. The less
the value ofD decreases (less).
Then the more efficient sterilization will be. This is because Dvalue is the
time required to destroy microorganisms. So less time (D value) results
in killing more bacteria, so sterilization becomes very efficient.
i Z value:
This represents the amount of temperature change required to
decrease one point in D- value.
Change in temperature is the Z- value
As the temperature rises, more bacteria are killed and the D- value
decreases and the Z- value increases.
As Z value is proportional to the D- value and a lower D value will
result ina more efficient sterilization.
F- yalue:
Eficacy of asterilization method is determined by the number of minutes
it takes to kill bacterial spores through heating. It is possible to
calculate the probability of survivors remaining from a load using the F
values follows;

[F=D(log No - log N)]

Where, TenpeZvae

No = initial population volume original population number

N= number of final residents volume ofunits
D= Dthe organism's temperature at 121 degree Celsius.
 DEQUIPMENTSEMPLOYED IN LARGE SCALE
STERILIZATION
List of equipment's employed in large scale sterilization:
1. Autoclave
2. Hotair oven

3. Microwave

4. HEPA filter

1.AUTOCLAVE
> Principle
The higher pressure also ensures the rapid penetration of heat into the
deeper parts of equipment. The moisture present in the steam causes
coagulation of proteins of microbes causing irreversible loss of their
activity and functions
> Construction
The autoclave is made of following components
Pryore contrel
1. Vessel or pressure chamber
2. Lid or door
3. Pressure gauge
neM
4. Pressure releasing unit (whistle)
5. Safety valve
6. Electrical heater
Peertd

The laboratory autoclave or pressure cooker

type autoclave consists of a vertical or
horizontal cylinder of gun metal Or
stainless steel in a supporting frame or case.
 The lid is fastened by screw clamps and rendered airtight by a asbestos
gasket.
The autoclave has on its lid or upper side a discharge tap for air and
steam a pressure gauge and a safety valve that can be set to blow off at
any desired pressure.
> Working
Stens inAutocave vcle
1. Boiling phase: The electric heat causes boiling of water and generate the
steam. The produced steam replaces the trapped air by displacement.
2. Rising temperature phase: The temperature rises and reaches up to the
set level i.e. 121°C.

3. Sterilization time: This is the time when microbes are killed.

4. Release the pressure: The entrapped pressure is released by opening

the valve.

O The steam circulates within the jacket & is supplied under high
pressure to closed inner chamber where articles for sterilization are kept.

O One fifth of the cylinder is filledwith water, materials to be sterilized are

kept inside, lid closed & heater is put on. Safety valve is adjusted to
required pressure.

OThe boiling of water inside the chamber after sometime results in steam
which is allowed to escape with air mixture fill the cylinder becomes air
free.

OThe discharge tap is closed and the desired pressure inside in chamber
is allowed to rise to the one chosen for autoclaving for a âxed time ls
thus complete sterilization achieved.
Standard operating procedures for autoclaving is: 115 C, half an hour. 121
°C, 15 minutes.
 S.NO TIME TEMPERATURE PRESSURE
in degree Celsius

1 80 min 110 O lbs

2 60 min 110 5 lbs

3 40 min 116 10 lbs
4 15 min 121 15 lbs
5 10 min 126 20 lbs

6 3 Min 130 25-30 1bs

Advantages
Rapid andeffective
Effective for sterilizing cloth surgical packs and towel packs.
" Less time consuming.
Applicable for both thermoplastic and thermolabile
components.

Disadvantages
"Items sensitive to heat cannot be sterilized
" It tends tocorrode carbon steel bum and instruments.
" It is costly technique.
>Applications
It is used to sterilize anything., which is not injured by steam and high
temperature of sterilization. These inchudes, Aqueous parenteral
solutions e.g. distilled water, saline solutions
Aqueous liquid media e.g. liquid media with or without carbohydrate
and gelatin.
" Surgical dressings and fabrics.
" Plastic and rubber closures.
" Metal instruments.

Glass apparatus and containers.

 2, HOTAIR OVEN
> Principle:_It kills microorganisms by destructive oxidation of
essential cell constituents.
Construction:
" The nmodern hot air ovens consist of a double walled chamber of
aluminium or stainless steel separated from the outer case by a thick
layer of insulation made of fiberglass. Thermometer
" Insulation is also filled in the hollow Double-walled
flanged door which carries an insulated
chamber
asbestos jacket that provides a Aluminium
tight seal. tray

Heating is affected by electrical

Asbestos
heating elements and thermo-stats oor jacket
automatically control
Timer 4Main on or
temperature. oi suitch
Temperature Direct Heat reulotor
regulator knob hrough konob
Working timer

Oven is an electrical devise that works on Heat convection principle. It

consist of electrical heating coil that produces dry heat. In the chamber, the
produced dry heat air displaces the cooled air forming heat gradient.
The use of fan allows to the uniform distribution ofheat.

S. NO. TEMPERATURE TIME

170°C 30 Min

2 160°C 60 Min

3 150°C 150 Min

> Application
" Used to sterilize the equipment's like glassware, forceps, scissors,
spatula, swabs, some pharmaceutical substances such as glycerin, fixed
oil, paraffin propylene.
" Used in many industries for drying and baking and curing process.
" Used to sterilize powders and other non volatile compounds.
> Advantages
1.It is Eco-friendly
2.It is most efficient method to degrade microbial endotoxins.
3.It is safer than autoclave.

4.It is the ideal instrument for sterilizing oil and powders.

Disadvantages
1. It is time consuming because the dry heat penetrate slowBy as
compared to moistheat.
2. It may not be efficient to degrade prions.
3. The heat sensitive or heat labile material cannot be sterilized.

4. It cannot be operated without electricity.

3. HEPA FILTER
Air purifiers reduce air pollutants by removing particulate matter (HEPA), or
High-Efficiency Particulate Absorbers. An example of a filter with 99.97
percent efficiency would be one that can trap particles smaller than 93
microns. During the pass of a HEPA filter, particdes are emitted in four
directions:
1. Direct impaction - large contaminants like dust,
mold, pollen, and mold adhere to fiber In a
straight line and travel directly through It.
2. Sieving - When an air particle moves between two
fibers, ifs bigger than the gaps, and thus becomes
ensued .
 3. Interception -The air is capable of rerouting around fibers, but due to
inertia, particles continue on their path and remain attached to the fibers.
4. Diffusion - It is more likely that ultrafine particles willhit and stick to
fibers because they move more erratically than larger ones.
norte inpetto teteception

Dimeken

nd eCe

ADVANTAGES
" In addition to sterilizing thermolabile medications, such as blood
products, insulin, and enzymes, the method is also suitable for sterilizing
blood plasma.
During the preparation, all types of bacteria are removed including those
that are alive and those that are dead.

The sterilization process is performed simultaneously with the

clarification process.
A parenteral solution in a small quantity can be rapidly supplied in an
emergency with this method.
DISADVANTAGES

" Sterility testing is necessary because the method is not reliable.

"With this method, it is not possible to sterilize suspensions and oily
preparations.
Filters may allow medicaments to be absorbed from a solution.
There are no immediate indications when the media is defeçtive. Aseptic
techniques must be used.
" Staff must be highly trained.
> APPLICATIONS
Parenteral solution containing thermolabile medicines can be sterilized
using this method without decomposition,such as insulin, blood stream,
and other products containing protein matter, such as heat sensitive
injections and biological products.

|OSTERLITY INDICATORS
It is essential that strict controls are carried out on products to be labeled
'sterile. Such controls must then ensure, the absence of viable
microorganisms from these products.
There are basically two types of controls:
1. Controls on the process of sterilization i.e. sterilization monitors or
sterilization indicators.

2. Sterility testingof the products.

3. Monitoring of the sterilization process can be achieved by the use of
physical, chemical or biological indicators of the sterilization
performance.
PHYSICAL INDICATORS
1. Moist heat:
A master process record (MPR) is prepared as part of the validation
procedure for a particular autoclave and for each specified product and load
configuration. The MPR should be checked at annual intervals and whenever
significant changes occur in the BPR when compared with the MPR.
2. Dry heat:
In dry heat sterilization processes, a temperature
record chart made of each sterilization cycle
and is compared against a master temperature
record.
 3. GaseOuS:
For gaseous sterilization procedures, elevated temperatures are
monitored for each sterilization cycle by temperature probes and
routine leak tests are performed to ensure gas-tight seals.
Gas concentration is measured independently of pressure
rise,often by reference to the weight of gas used. Pressure
and humidity measurements are recorded.
4. Filtration:
" Bubble point pressure test is a technique employed for determining the
pore size of filters and may also be used to check the integrity of certain
types of filter devices immediately after use.
" The principle of the test is that the filter is soaked in an
appropriate fluid and pressure is applied to the filter.
The pressure difference when the first bubble of air breaks away from
the filter is equivalent to the maximum pore size. When the air
pressure is further increased slowly, there is general eruption of bubbles
over the entire surface. The pressure difference is equivalent to the
mean pore size.

> CHEMICAL INDICATORS

Chemical monitoring of a sterilization process is based on the ability of heat,
steam, sterilant gases and ionizing radiation to alter the chemical or
physical characteristicsof a variety of chemical substances.
1Browne tubes
Each tube consists of a sealed glass tube which contains a red fluid (an
ester andacid- base indicator) that changes to yellow, brown and finally
green on heating(the ester undergoes heat hydrolysis to form an acid
alcohol. The acid will change the color of the indicator.
 2.Witness tubes:
Witness tubes consist of single crystalline substances of known melting
point contained in glass tubes e.g. Sulphur (115°C), succinic anhydride
(120°C), benzoic acid (121°C) etc. A dye may be included to show more
clearty that the crystals have melted.
Such a device only indicates that a certain temperature has been
reached. Exposure time can be calculated by putting the crystals in one
end of an 'hour-glass' tube, the volume of the crystals and the diameter
of the constriction of the tube being adjusted so that the time for transfer
of the melt is the same as that required for the sterilization at the
required temperature
3. Royce sachet:
The Royce sachet is a chemical indicates for ethylene oxide
sterilization. This consists of a polythene sachet containing
magnesium chloride, HCI and a bromophenol blue indicator. Agiven
concentration-time exposure to ethylene oxide results in the formation
of ethylene chlorohydrin and a color change from yellow to purple.
BIOLOGICAL INDICATORS
1. Biological indicators consist of a suitable organism deposited on a
carrier and are distributed throughout the sterilizer load.
2. At the end of the sterilization process, the units are recovered and cultured
to determine the presence or absence of survivors. The biological
indicator measures sterilization processes directly and is able to integrate
all sterilization parameters.

3. The selected organism should possess high and reproducible resistance

to the sterilizing agent, should be genetically stable, readily
characterizable and non-pathogenic.
4. The viability of the organisms, the storage conditions before use and the
incubation and culture conditions after sterilization must be standardized
for the results. The organisms used as biological indicators are usually
resistant bacterial spores.