Exp. No: 1 Date: INTRODUCTION, LABORATORY SAFETY, USE OF EQUIPMENT, STERILIZATION TECHNIQUES CULTURE MEDIA ! TYPES AND USE PREPARATION OF NUTRIENT BROTH AND A"AR
a# "ENERAL "ENERA L LABORATORY SAFETY PROCEDURES PROCEDUR ES
Make sure to read the laboratory exercise before class and plan your work. This creates awareness of the special safety concerns for the laboratory class and permits efficient use of class time $ea% &a'o%ato%( )oat* a+ t-e+ e+te% t-e &a'o%ato%(.
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Wear We ar closed footwear to protect the feet. Long hair should be tied back.
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Keep all bags in the racks provided inside the lab
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Eating drinking smoking handling contact lenses applying cosmetics and storing food for human use are not permitted in the work areas.
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Do +ot 'e/+ a+( expe%/0e+ta& o%2 /t-o3t p%/o% o%/e+tat/o+ '( t-e /+*t%3)to%. $a*- (o3% -a+* t-o%o3-&( /t- *oap a+ ate% 'e4o%e *ta%t/+ a+( expe%/0e+t. Mo3t- p/pett/+ /* p%o-/'/te. !se mechanical devices for pipetting
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"roken glassware must not be handled directly by hand but must be removed by mechanical means such as a brush and dustpan tongs or forceps.
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#pills and accidents should be reported to the instructor
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$f a piece of e%uipment fails to work report it immediately to the lab instructor.
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&lean up the work place and replace all reagents in designated place before leaving the laboratory
b' MICROBIOLO"Y SAFETY PROCEDURES (ollow the general guidelines and prepare for experimentation. •
Keep your workbench neat and organi)ed for the experiment
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Wear We ar disposable latex gloves while handling blood products products *e.g. whole blood plasma serum' or cultures C&ea+ *&/e* )a%e43&&( a+ /pe /t /t- a&)o-o& 4o% 0/)%o*)op/) o%2. Label all cultures and solutions properly with with the name of the test organism the the name of
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the medium dilution of the sample your name or initials date course + lab section prior to inoculation Keep culture tubes on test tube racks when not in use and carry them in racks. ,rocedures should be performed carefully to avoid splashes or aerosols. $f a bacterial culture splashed in your eye*s' or on your skin immediately flush with
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copious amount of running water I4 a )3&t3% )3&t3%ee /* *p/&&e *p/&&e, , )o5e% )o5e% t-e *p/&&e *p/&&e 0ate 0ate%/a %/a&& /t/t- pape% pape% toe&* toe&* a+ a+ app& app&( (
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&a'o%ato%( /*/+4e)ta+t *3)- a* 16 *o/30 -(po)-&o%/te *o&3t/o+ o% 786 et-a+o& o5e% t-e *p/&& a%ea. Keep the towel on the spill for - minutes. /isposable gloves should be worn while cleaning spills. $nform your instructor of the spill. ,lace the towel in an autoclave waste bag provided. Ensure you wash your hands immediately after dealing with the spill. Working with hot items either from the autoclave or heated in the "unsen burner re%uires
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protection of your hands. Wear Wear protective gloves or handle the hot item with tongs. Ne5e% &ea5e a &/-te B3+*e+ '3%+e% 3+atte+e. 0 fire extinguisher is ready in each laboratory. $f your clothes catch on fire 1drop and
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roll1 roll1 to smother the flames. flames. 2our 2our lab partners partners should use a fire blanket or their coats to help smother the flames.
Te%0/+at/o+ o4 *e**/o+* •
&lean up your bench as you work disposing used items properly.
b' MICROBIOLO"Y SAFETY PROCEDURES (ollow the general guidelines and prepare for experimentation. •
Keep your workbench neat and organi)ed for the experiment
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Wear We ar disposable latex gloves while handling blood products products *e.g. whole blood plasma serum' or cultures C&ea+ *&/e* )a%e43&&( a+ /pe /t /t- a&)o-o& 4o% 0/)%o*)op/) o%2. Label all cultures and solutions properly with with the name of the test organism the the name of
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the medium dilution of the sample your name or initials date course + lab section prior to inoculation Keep culture tubes on test tube racks when not in use and carry them in racks. ,rocedures should be performed carefully to avoid splashes or aerosols. $f a bacterial culture splashed in your eye*s' or on your skin immediately flush with
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copious amount of running water I4 a )3&t3% )3&t3%ee /* *p/&&e *p/&&e, , )o5e% )o5e% t-e *p/&&e *p/&&e 0ate 0ate%/a %/a&& /t/t- pape% pape% toe&* toe&* a+ a+ app& app&( (
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&a'o%ato%( /*/+4e)ta+t *3)- a* 16 *o/30 -(po)-&o%/te *o&3t/o+ o% 786 et-a+o& o5e% t-e *p/&& a%ea. Keep the towel on the spill for - minutes. /isposable gloves should be worn while cleaning spills. $nform your instructor of the spill. ,lace the towel in an autoclave waste bag provided. Ensure you wash your hands immediately after dealing with the spill. Working with hot items either from the autoclave or heated in the "unsen burner re%uires
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protection of your hands. Wear Wear protective gloves or handle the hot item with tongs. Ne5e% &ea5e a &/-te B3+*e+ '3%+e% 3+atte+e. 0 fire extinguisher is ready in each laboratory. $f your clothes catch on fire 1drop and
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roll1 roll1 to smother the flames. flames. 2our 2our lab partners partners should use a fire blanket or their coats to help smother the flames.
Te%0/+at/o+ o4 *e**/o+* •
&lean up your bench as you work disposing used items properly.
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,lace used glass slides and coverslips in glass dishes of disinfectant.
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0ll materials re%uiring incubation or refrigeration must be appropriately labelled and placed on the trays provided. T3%+ o4 a&& e93/p0e+t a4te% 3*e a+ %eae+t* a+ *3pp&/e* 03*t 'e %et3%+e to t-e/% e*/+ate p&a)e* 'e4o%e &ea5/+ t-e &a'o%ato%(.
Ste%/&/*at/o+ a+ /*po*a& /o not throw any bacterial culture in the sink. /o not dispose of any solid material in the • •
sink. 0ll cultures stocks and other regulated wastes are decontaminated before disposal by an
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approved approved decontam decontaminat ination ion method method such such as autocl autoclavi aving. ng. /ilute /ilute the cultur culturee with with 3 M sodium hydroxide before autoclaving and disposal. ,lace items that re%uire decontamination by autoclaving including flasks beakers and
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other containers in a cart. ,lace glass tube at an angle in baskets to avoid spillage. The caps of all screw4topped bottles must be loosened before cultures c ultures and media are sterilised. $t is very important that instructions for use of the auto clave are followed in order to achieve and maintain sufficiently high temperatures for a long enough time.
)# USE OF EQUIPMENT EQUIPM ENT AND STERILIZATION STERIL IZATION TECHNIQUE TECHNIQ UE IN MICROBIOLO"Y LABORATORY 1. C3&t3%e t3'e* a+ Pet%/ /*-e*:
5lass test tubes and glass or plastic ,etri dishes are used to cultivate microorganisms. 0 suitable nutrient medium in the form of broth *li%uid medium' or agar *solid medium' may be added to the culture tubes while only a solid medium is used in ,etri dishes. #terile environment is mainta maintaine ined d in cultur culturee tubes tubes by closin closing g the tubes with with non absorb absorbent ent cotton plugs. plugs. The necessary movements of air in and gaseous products out are not prevented by using cotton plugs. ,etri dishes provide a larger surface area for growth and cultivation. $t consist of bottom dish portion contains medium and larger top portion as a loose cover. (or routine purposes dishes approximately 36cm in diameter are used. The sterile agar medium of 36 to -ml is dispensed to previously sterili)ed dishes. A4te% /+o)3&at/o+ t-e Pet%/ /*- *-o3& 'e p&a)e /+ a+ /+5e%te po*/t/o+ to prevent condensation that forms on during solidification of agar. . E93/p0e+t* 4o% *te%/&/;at/o+: #terili)ation is the process of destroying all forms of microbial life. &ommon methods used for sterili)ation is outlined below
A. B3+*e+ '3%+e% 0 B3+*e+ '3%+e% named after 7obert "unsen is a common piece of laboratory e%uipment that produces a single open gas flame which is used for heating sterili)ation and combustion. The gas gas can can be natu natura rall gas gas *whi *which ch is mainl mainly y metha methane' ne' or a li%ue li%uefi fied ed petr petrol oleu eum m gas gas such such as propane butane or a mixture of both.
$t is used for sterili)ation of wire loops and *with alcohol' metal forceps and glass spreaders. B. A3to)&a5e $t is used for sterili)ing media solutions discarded cultures and contaminated materials. 0utoclave uses moist heat steam under pressure for inhibiting or destroying microorganisms. #team under pressure provides temperatures above those obtainable by boiling. boiling. 0utoclave is a double48acketed steam chamber e%uipped with devices which permit the chamber to be filled with saturated steam and maintained at a designated temperature and pressure for any period of time. /uring operation the chamber should be completely replaced by saturated steam. 5enerally autoclave is operated at a pressure of approximately 36lb+in - at 3-39&. Time re%uired to achieve sterility depends on the material to be sterili)ed type of the container and the volume. (or media and glass wares -minutes - minutes is re%uired for efficient sterili)ation C. Hot a/% o5e+ $t is recommended when exposure of materials to moist heat is undesirable. $t contains rectangular chamber made up of double walls with insulating material between the wall spaces. :ot air oven uses electric coils or gases to heat the chamber. (or laboratory glass wares -hr exposure to a temperature of 3;9& is sufficient for sterili)ation. D. F/&te%* $t is used to remove microorganisms from li%uids or gases. H/- E44/)/e+)( Pa%t/)3&ate A/% 4/&te%*
ability to penetrate matter. Even a thin layer of glass filters off a large percentage of light. Thus only the microorganisms on the surface of the ob8ect are susceptible for destruction.
=. Mate%/a&* 4o% t%a+*4e%%/+ 0/)%o'/a& )3&t3%e*: Microorganisms must be transferred from one vessel to another or from stock cultures to various media for maintenance. $t is called *3')3&t3%/+ and must be carried out under sterile conditions to prevent contamination.
A. M/)%o P/pette*: !sed for handling small amount of volume from 3ml to 3>l. There are two types of pipettes 0ir displacement pipette and positive displacement pipette. 0ir displacement pipettes are meant for general use with a%ueous solutions. ,ositive displacement pipettes are used for high viscosity and volatile li%uids. B. $/%e &oop* a+ +ee&e*: Made of nichrome or platinum. $t is extremely durable and is easily sterili)ed by incineration using flame from "unsen burner. $t is used for techni%ues such as streak plating and for preparation of stab cultures.
(ig? a' $noculation needle b' $noculation loop Wire loops are sterili)ed using red heat in a "unsen flame before and after use. They must be heated to red hot to make sure that any contaminating bacterial spores are destroyed. The handle of the wire loop is held close to the top. This leaves the little finger free to take hold of the cotton wool plug+ screw cap of a test tube+bottle.
>. C3&t/5at/o+ )-a0'e%*: Microorganism should be grown at their optimum temperature. I+)3'ato% is used to maintain temperature during the necessary growth period. $t is an insulated metallic chamber and is divided into compartments by metallic racks to hold test tubes and ,etri dishes. $ncubator uses dry heat and is thermostatically controlled so that temperature can be varied depending on the re%uirements of specific microorganisms. I+)3'ato% /t- *-a2e% provides increased aeration by agitating the vessel. $t can be used only for cultivation of organisms in li%uid medium. ?. Re4%/e%ato%: !sed for maintenance and storage of stock cultures samples and chemicals at a temperature between 9& to @9&. $n low temperature bacteria shows no metabolic activity and
there will be no growth of microorganisms. Thus refrigeration is bacteriostatic. /eep free)er *4 -9& and 4A9&' is used for long term storage of stock cultures isolated /B0 7B0 ,roteins and en)ymes. #tock cultures are stored upon addition of glycerol to maintain the cells in viable condition. @. M/)%oa5e o5e+ 0 microwave oven is used to melt microbiological media resulting in a substantial reduction of heat generation and considerable savings in time. # CULTURE MEDIA ! TYPES AND USES PREPARATION OF MEDIA AIM: To prepare nutrient agar and nutrient broth medium for growth of microorganisms PRINCIPLE:
The survival and growth of microorganisms depends on the ade%uate supply of nutrients and a favorable growth environment. 0 culture medium may be classified by three ways based on consistency nutritional composition and application. /. C&a**/4/)at/o+ 'a*e o+ )o+*/*te+)(: &ulture media are solid li%uid or semisolid. 0 li%uid medium which lacks a solidifying agent is called '%ot- 0e/30. 0 broth medium supplemented with solidifying agent like agar results in semisolid or solid medium. 0gar is an extract of seaweedC a complex carbohydrate composed mainly of galactose and it does not contribute any nutritive property as most of the bacteria cannot hydroly)e agar. 0gar is an excellent solidifying agent as it li%uefies at 39& and solidifies at @9&. Thus microorganisms can be grown at D=9& and slightly above without li%uefaction of medium. Most commonly 34D of agar is used for solid medium. &oncentration below this *.-4.6' is used for semi4solid medium. //. C&a**/4/)at/o+ 'a*e o+ )o0po*/t/o+: C-e0/)a&&( e4/+e 0e/a: $t composed of pure ingredients in carefully measured concentrations dissolved in double distilled water i.e. the exact chemical composition of the medium is known. Typically they contain a simple sugar as the carbon and energy source an inorganic nitrogen source various mineral salts and if necessary growth factors *purified amino acids vitamins purines and pyrimidines'. Co0p&ex 0e/a: &omplex media are rich in nutrients they contain water soluble extracts of plant or animal tissue *e.g. en)ymatically digested animal proteins such as peptone and tryptone'. !sually a sugar often glucose is added to serve as the main carbon and energy source. The combination of extracts and sugar creates a medium which is rich in minerals and organic nutrients but since the exact composition is unknown the medium is called complex. ///. C&a**/4/)at/o+ 'a*e o+ app&/)at/o+:
Se&e)t/5e 0e/a: $t supports the growth of only certain types of bacteria. Media can be made selective through the addition of substances that enhance or inhibit the growth of particular types of bacteria. Ex? Mac&onkey 0gar4 selective for gram negative bacteria D/44e%e+t/a& 0e/a: $t reveals specific metabolic or metabolic characteristics of bacteria grown on it. &ertain reagents or supplements when incorporated into culture media allow differentiation of various kinds of bacteria based on their colony color. Ex? Mac&onkey agar contains neutral red *p: indicator' helps to differentiate lactose fermenting bacteria. E+%/)-e 0e/a:
,romotes the growth of a particular organism by providing it with the
essential nutrients and rarely contains inhibitory substances to prevent the growth of normal competitors Me/a
P3%po*e
#elective
#uppress unwanted microbes or encourage desired microbes
/ifferential /istinguish colonies of specific microbes from others Enrichment #imilar to selective media but designed to increase the numbers of desired microorganisms to a detectable level without stimulating the rest of the bacterial population MATERIALS REQUIRED ? Media components conical flask p: meter /istilled water Test tubes &otton ,etri plates 0utoclave ,aper PROCEDURE: N3t%/e+t '%ot- )o0po*/t/o+: for 36ml ,eptone4 3.6g #odium chloride4 .=g 2east extract4 .@6g 3. Weigh re%uired components and transfer to -6ml conical flask. Make up the volume to 3ml using distilled water. -. 0d8ust p: to =.D using .3M BaF: D. Make up the volume to 36ml and check p: again @. ,lug the flask with cotton and wrap it with paper. 6. 0utoclave at 36,si for -min N3t%/e+t aa% )o0po*/t/o+: for 3ml ,eptone43g #odium chloride4.6g 2east extract4.Dg 0gar4-g
3. Weigh re%uired components and transfer to -6ml conical flask. Make up the volume to 3ml using distilled water. -. 0d8ust p: to =.D using .3M BaF: D. Make up the volume to 3ml and check p: again @. Weigh and add -g of agar. 6. ,lug the flask with cotton and wrap it with paper. 0utoclave at 36,si for -min RESULT:
EP: DATE: CULTURE TECHNIQUES, ISOLATION AND PRESERATION OF CULTURES ! BROTH: FLAS, TEST TUBES SOLID: POUR PLATES, STREA PLATES, SLANTS, STABS
a# ISOLATION OF PURE CULTURES STREA PLATE METHOD AIM: To perform streak plate procedure for isolation of single colony from a mixed culture PRINCIPLE:
$n nature microorganisms exist as mixed population in widely differing types. :owever to obtain the knowledge of particular type of microorganisms it is essential to separate or isolate these organisms from the mixed population.
Re*3&t:
Co+t/+3o3* *t%ea2/+: 3. (lame and cool the loop. Take loopful of mixed culture on the agar surface. -. /rag the inoculation loop on the agar surface continuously from left to right as shown in figure.
RESULT:
'# INOCULATION OF NUTRIENT BROTH, NUTRIENT A"AR SLANTS, STABS AIM: To inoculate isolated colony from streak plate in nutrient broth nutrient agar slants and stabs PRINCIPLE:
Fnce discrete colonies develop on the surface of agar plate each colony may be picked up from agar plate and grown on nutrient broth agar or slants. Each of these cultures represents pure or stock culture and can be used to study cultural characteristics of microorganisms. APPARATUS REQUIRED: $noculation loop inoculation needle Butrient agar slant Butrient agar stab PROCEDURE: A. I+o)3&at/o+ o4 aa% *&a+t*: 3. &lean the laminar hood and light the burner and place the re%uired materials inside the laminar hood.
-. (lame the inoculation loop until it becomes red. D. &ool the flame for 3seconds. 0 hot loop will damage the bacteria cells. ,ick single colony from streak plate @. !ncap agar slant culture and show mouth of the tube in flame. 6. $noculate the culture by drawing the loop over the surface of the agar in )ig)ag motion. &are should be taken not to dig the agar slant. ;. 7eflame the inoculation loop and mouth of the tube. ,lug tube with cotton. =. $ncubate the tube at D=9& in the incubator for overnight for the growth of pure culture
B. I+o)3&at/o+ o4 aa% *ta'*: 3. (lame the inoculation needle and pick single colony from streak plate -. !ncap the culture tube containing agar and show mouth of the tube in flame. D. $nsert the needle to the bottom of the tube through the agar and withdraw along the line of insertion @. 7eflame the inoculation needle and mouth of the tube. ,lug tube with cotton 6. $ncubate the tube at D=9& in the incubator for overnight C. I+o)3&at/o+ /+to +3t%/e+t '%ot- 0e/30 3. #terili)e the inoculation loop and pick single colony from streak4plate -. (lame the mouth of the culture tube and inoculate into nutrient broth by dislodging the inoculum from the loop by slight agitation+ rotation in the broth D. 7eflame the inoculation loop and mouth of the tube. ,lug tube with cotton @. $ncubate the tube at D=9& in the incubator
RESULT:
EP: = DATE: MICROSCOPY $ORIN" AND CARE OF MICROSCOPE AIM: 3. To identify all the parts of a compound microscope -. Know how to use the microscope and oil immersion lens
MATERIALS REQUIRED: &ompound microscope immersion oil lens cleaner glass slide cover slip THEORY AND PRINCIPLE: The magnification of small things is a necessary facet of biological research but the fine
detail in cells and in subcellular components re%uires that any imaging system be capable of providing spatial information across small distances. 7esolution is defined as the ability to distinguish two very small and closely4spaced ob8ects as separate entities. 7esolution is best when the distance separating the two tiny ob8ects is small. 7esolution is determined by certain physical parameters that include the wavelength of light and the light4gathering power of the ob8ective and condenser lenses. 0 simple mathematical e%uation defines the smallest distance *dmin' separating the two very small ob8ects? 0/+ 1. x a5e&e+t- N.A.
N.A. )o+e+*e%
o'Ge)t/5e
This is the theoretical resolving power of a light microscope. $n practice specimen %uality usually limits dmin to something greater than its theoretical lower limit. B.0. *Bumerical 0perture' is a mathematical calculation of the light4gathering capabilities of a lens. The B.0. of each ob8ective lens is inscribed in the metal tube and ranges from .-643.@. The higher the B.0. the better the light4gathering properties of the lens and the better the resolution. :igher B.0. values also mean shorter working distances *you have to get the lens closer to the ob8ect'. B.0. values above 3. also indicate that the lens is used with some immersion fluid such as immersion oil.
(rom the e%uation above you should be aware that the B.0. of the condenser is as important as the B.0. of the ob8ective lens in determining resolution. $t is for this reason that closure of the condenser diaphragm results in a loss of resolution. $n practice at full aperture and with good oil immersion lenses *B.0. 3.@ for both the condenser and the ob8ective' it is possible to be able to resolve slightly better than .- >m. (rom the e%uation above it should also be clear that shorter wavelength light *bluer light' will provide you with better resolution *smaller dmin values'. :owever there are practical considerations in how short the wavelength can be. $n the early 3G6Is a !< microscope was designed but re%uired %uart) ob8ectives and a speciali)ed imaging device. The %uart) lenses provided slightly better resolution *dmin J .3 >m' but image %uality suffered from an inability on the part of the manufacturers to correct for aberrations caused by the %uart). The human eye is best adapted for green light and our ability to see detail may be compromised somewhat with the use of blue or violet. Most manufacturers of microscopes correct their simplest lenses *achromats' for green light.
- Magnification and Imaging Most microscopes in current use are known as compound microscopes where a magnified image of an ob8ect is produced by the ob8ective lens and this image is magnified by a second lens system *the ocular or eyepiece' for viewing. Thus final magnification of the microscope is dependent on the magnifying power of the ob8ective times the magnifying power of the ocular. Fb8ective magnification powers range from @ to 3. Lower magnification is impractical on a compound microscope stand because of spatial constraints with image correction and illumination. :igher magnification is impractical because of limitations in light gathering ability and shortness of working distances re%uired for very strong lenses. Fcular magnification ranges are typically A43- though 3 oculars are most common. 0s a result a standard microscope will provide you with a final magnification range o f @ up to 3.
Co0po+e+t* o4 0/)%o*)ope: 1. O'Ge)t/5e: $ts basic function is to gather the light passing through the specimen and then to pro8ect • •
an accurate real inverted $M05E of the specimen up into the body of the microscope. The ob8ective must be constructed so that it will be focused close enough to the specimen
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so that it will pro8ect a magnified real image up into the microscope. The higher power ob8ectives should have a retractable front lens housing to protect the
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front lens where the ob8ective re%uires focusing very close to the specimen. To the extent possible corrections for lens errors *aberrations' should be made within the
ob8ective . E(ep/e)e o% O)3&a%*: $ts basic function is to look atN the focused magnified real image pro8ected by the •
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ob8ective and magnify that image a second time as a virtual image seen as if 3inches from the eye. The eyepiece houses a fixed diaphragm. $t is at the plane of that fixed diaphragm that the
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image pro8ected by the ob8ective will be seenN Fn the shelf of the fixed diaphragm the eyepiece can be fitted with scales or markers or pointers or crosshairs that will be in simultaneous focus with the focused image
=. S3'*tae )o+e+*e%: $ts basic function is to gather the light coming from the light source and to concentrate • •
that light in a collection of parallel beams onto the specimen. The light gathered by the condenser comes to a focus at the back focal plane of the
ob8ective Ot-e% )o0po+e+t* ? The base of the microscope contains a )o&&e)to% &e+*. This lens is placed in front of the •
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light source. $ts function is to pro8ect an image of the light source onto the plane of the condenserHs aperture diaphragm. $n some instruments a diffusion or frosted filter is placed 8ust after the collector lens *side closer to the specimen' in order to provide more even illumination. 0lso in the base of the microscope under the condenser is a 4/%*t *3%4a)e 0/%%o%
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*silvered on its front surface only'. $ts function is to reflect the light coming from the lamp up into the substage condenser. 0t the lowest part of the observation tubes *binocular or trinocular' there is incorporated
a t3'e &e+*. $ts function is to gather the parallel rays of light pro8ected by the ob8ective *in infinity4corrected systems' and bring those rays to focus at the plane of the fixed diaphragm of the eyepiece. $n the instruments of some manufacturers the tube lens is built into the body of the microscope itself. Me)-a+/)a& E&e)t%/)a& )o0po+e+t*:
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The stand of the microscope houses the mechanical+electrical parts of the microscope. $t
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provides a sturdy vibration4resistant base for the various attachments. The base of the Flympus microscopes is 24shaped for great stability. $t houses the
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electrical components for operating and controlling the intensity of the lamp. The lamp may be placed depending on the instrument at the lower rear of the stand or directly under the condenser fitting. The base also houses the variable field diaphragm. The base may also have built in filters and a special circuit for illumination intensity for photomicrography. "uilt into the stand is a fitting to receive the microscope stage. The stage has an opening
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for passing the light. The specimen is placed on top of the stage and held in place by a specimen holder. 0ttached to the stage are concentric 42 control knobs which move the specimen
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forward +back or left+right. Fn the lower right and left side of the stand are the concentric coarse and fine focusing
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knobs. These raise or lower the stage in larger + smaller increments to bring the specimen into focus. 0bove the stage the stand has a nosepiece *may be fixed or removable' for holding the
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ob8ectives of various magnifications. The rotation of the nosepiece can bring any one of the attached ob8ectives into the light path *optical axis'. The nosepiece may also have a slot for special attachments. 7emovable observation tubes either binocular or trinocular are attached to the stand above the nosepiece. The binocular is used for viewing and the trinocular is used for viewing and +or photography. The observation tubes are usually set at approximately a D degree angle for comfortable viewing and may be tiltable or telescoping push4pull for greater flexibility.
EP: > DATE: IDENTIFICATION OF MICRO OR"ANISMS: STAININ" TECHNIQUES ! SIMPLE STAININ" AIM:
To prepare and stain bacterial smears made from broth and solid media and evaluate cell morphology. PRINCIPLE: The development of staining techni%ues was of great importance to microbiology. #ince many bacteria do not have pigments it can be difficult to see individual cells under a light *bright4field' microscope. #tains enhance the contrast and allow the microscopist to view the cell more distinctly. #taining not only makes bacteria more easily seen but it allows their morphology *e.g. si)e and shape' to be visuali)ed more easily. #tains range from simple to complex. #imple stains involve only one reagent and stain all bacteria similarly. They are useful solely for increasing contrast so that morphology si)e and arrangement of organisms can be determined. More complex stains involve multiple reagents and are often differential. 0 differential stain /*p&a(* t-e )-e0/)a& /44e%e+)e* in cellular structures including the cell wall and cell membrane because the macromolecules within the structure bind to different components of the stain. This means that they stain different types of bacteria differently. $n some cases specific stains can be used to visuali)e certain structures *flagella capsules endospores etc' of bacterial cells. #taining is based on the principle that opposite charges attract and that like charges repel. Most bacteria when placed in an a%ueous environment with the p: at about = have a net electrical charge that is negative. These negatively charged cells will attract positively charged molecules and repel those molecules that are negative. #tains *dyes' are chemicals containing chromophores groups that impart color. Their specificity is determined by their chemical structure. #tains are generally salts in which one of the ions is colored. *0 salt is a compound
composed of a positively charged ion and a negatively charged ion.' $n most commonly used dyes *basic dyes' the cation is the chromophore. "asic dyes include methylene blue crystal violet and safranin. These are used to prepare a simple stain. (or example the dye methylene blue is actually the salt methylene blue chloride which will dissociate in water into a positively charged methylene blue ion which is blue in color and a negatively charged chloride ion which is colorless. &ommonly used microbiological stains generally fall into one of two categories 4 basic stains or acidic stains *although there are a few stains such as $ndia $nk' which are neutral'. 0 basic dye is a stain that is cationic *positively charged' and will therefore react with material that is negatively charged. The cytoplasm of all bacterial cells have a slight negative charge when growing in a medium of near neutral p: and will therefore attract and bind with basic dyes. #ome examples of basic dyes are crystal violet safranin basic fuchsin and methylene blue. 0cid dyes have negatively charged chromophores and are repelled by the bacterial surface forming a deposit around the organism. They stain the background and leave the microbe transparent. Bigrosine and congo red are examples of acid dyes. P%epa%/+ Sta/+*
When preparing a stain a perfectly clean microscope slide must be used. Bew slides are usually the best however if used slides are used great care should be taken to clean all greasy film from the slide. &leanliness can be tested by dropping a drop of water on the slide. $f it spreads over the entire slide the slide is clean. 0ny beading of the water indicates the presence of a greasy film. 0 thin film of bacteria should be spread upon the slide. $f the smear is too thick it is difficult to see anything because there will be little light passing through. The smear should be thin and allowed to dry. Fnce the smear has dried the slide should be passed over a lit "unsen burner several times to affix the organisms. This procedure is known as heat fixing. There is a slight shrinkage of cells during this process which is normal but it helps the bacterial cells to adhere to the slide through several rinses. $f the slide is overheated the cells will warp and structure will be indistinguishable. $f heat is applied to the cell before the smear is dry there willbe distortion. 0 properly stained bacterial smear should be slightly difficult to see to the naked eye. $f there are dark splotches of color the bacteria are piled on top of each other. (inished stained smears will last for months stored in a cool dark place provided no oil is present on the stain. There are solvents such as xylol that can be used to remove excess oil from slides that are to be saved. #olvents however strip any markings made by wax pencils so re4 labeling is important.
Ba)te%/a& Mo%p-o&o(:
"acteria are very small unicellular microorganisms ubi%uitous in nature. They are micrometers *3>m J 34; m' in si)e. They have cell walls composed of peptidoglycan and reproduce by binary fission. "acteria vary in their morphological features. The most common morphologies are? •
&occus *pleural? &occi'?
#pherical bacteriaC may occur in pairs *diplococci' in groups of four *tetracocci' in grape4like clusters *#taphylococci' in chains *#treptococci' or in cubical arrangements of eight or more *sarcinae'. (or example? #taphylococcus aureus #treptococcus pyogenes •
"acillus *pleural? "acilli'?
7od4shaped bacteriaC generally occur singly but may occasionally be found in pairs *diplo4 bacilli' or chains *streptobacilli'. (or example? "acillus cereus &lostridium tetani •
#pirillum *pleural? #pirilla'
#piral4shaped bacteria (or example? #pirillum
#ome bacteria have other shapes such as?
&occobacilli? Elongated spherical or ovoid form. (ilamentous? "acilli that occur in long chains or threads. (usiform? "acilli with tapered ends. MATERIALS REQUIRED: Microscope slides &over slips $noculating loops "roth cultures of various bacteria Microscopes
D. 7esuspend the bacteria in the broth by rolling the tube between the hands. "acteria must always be resuspended before removing any inoculum. @. !sing aseptic techni%ues transfer a loop full of bacteria from the tube to the labelled circle on the slide. Keep the slide and the tube near the flame. 0void inhaling any aerosols. (lame the loop after transfer. 6. 0llow the smear to dry. ;. When the smear is completely dry pass the slide through the top of the "unsen burner flame several times to heat fix the organisms. =. Then proceed to ,rocedure 3. P%o)e3%e 1 S/0p&e Sta/+/+
3. ,lace the slides on the stain rack over the sink. -. &over the slides with one of the stains and allow the stain to stay on the slide for following intervals. 3. 3 &rystal violet 4 D seconds to 3 minute -. .3 "asic fuchsin 4 - to D minutes D. 3 LoefflerHs Methylene blue 4 - to D minutes @. .6 #affranin 4 3 minute D. :old the slide still tilted to the side and begin to rinse with deioni)edwater from the supplied water bottles. 0im around the smear and remove all excess stain. /o not aim right at the smear as it may result in the removal of the smear. @. #hake all excess water from the slide. 6. #lides can be air dried but to avoid any chance of decolori)ation by water you may blot the slides dry in the book of bibulous *absorbent' paper. ;. Examine the stained smears on the microscope. The smears should be examined on every power including the oil immersion lens. =. /raw what is seen in the field of view on the oil immersion lens below. Fnce done cleanup work area and dispose of gloves and slides in a bioha)ard bag. RESULT :
EP:? DATE: IDENTIFICATION OF MICRO OR"ANISMS: STAININ" TECHNIQUES ! "RAM STAININ" AIM: •
To differentiate between the two ma8or categories of bacteria? 5ram positive and 5ram
•
negative. To understand how the 5ram stain reaction affects 5ram positive and 5ram negative bacteria based on the biochemical and structural differences of their cell walls.
PRINCIPLE:
#taining is an auxiliary techni%ue used in microscopic techni%ues used to enhance the clarity of the microscopic image. #tains and dyes are widely used in the scientific field to highlight the structure of the biological specimens cells tissues etc. The most widely used staining procedure in microbiology is the 5ram stain discovered by the /anish scientist and physician :ans &hristian Ooachim 5ram in 3AA@. 5ram staining is a differential staining techni%ue that differentiates bacteria into two groups? gram4positives and gram4negatives. The procedure is based on the ability of microorganisms to retain color of the stains used during the gram stain reaction. 5ram4negative bacteria are decolori)ed by the alcohol losing the color of the primary stain purple. 5ram4positive bacteria are not decolori)ed by alcohol and will remain as purple. 0fter decolori)ation step a counter stain is used to impart a pink color to the decolori)ed gram4negative organisms. The 5ram stain procedure enables bacteria to retain color of the stains based on the differences in the chemical and physical properties of the cell wall. 3. 5ram positive bacteria? #tain dark purple due to retaining the primary dye called &rystal
&lean glass slides $noculating loop "unsen burner "ibulous paper Microscope Lens paper and lens cleaner $mmersion oil /istilled water 3A to -@ hour cultures of organisms REA"ENTS:
3. ,rimary #tain
4
&rystal
-. Mordant
4
5rams $odine
D. /ecolouri)er
4
Ethyl 0lcohol
@. #econdary #tain
4
#afranin
PROCEDURE: Pa%t 1: P%epa%at/o+ o4 t-e &a** 0/)%o*)op/) *&/e
5rease or oil free slides are essential for the preparation of microbial smears. 5rease or oil from the fingers on the slides is removed by washing the slides with soap and water. Wipe the slides with spirit or alcohol. 0fter cleaning dry the slides and place them on laboratory towels until ready for use. Pa%t : La'e&/+ o4 t-e *&/e*
/rawing a circle on the underside of the slide using a glassware4marking pen may be helpful to clearly designate the area in which you will prepare the smear. 2ou may also label the slide with the initials of the name of the organism on the edge of the slide. &are should be taken that the label should not be in contact with the staining reagents. Pa%t =: P%epa%at/o+ o4 t-e *0ea% •
Ba)te%/a& *3*pe+*/o+* /+ '%ot-: With a sterile cooled loop place a loopful of the broth
culture on the slide. #pread by means of circular motion of the inoculating loop to about one centimeter in diameter. Excessive spreading may result in disruption of cellular arrangement. 0 satisfactory smear will allow examination of the typical cellular arrangement and isolated cells. •
Ba)te%/a& p&ate )3&t3%e*: With a sterile cooled loop place a drop of sterile water or
saline solution on the slide. #terili)e and cool the loop again and pick up a very small sample of a bacterial colony and gently stir into the drop of water+saline on the slide to create an emulsion.
•
Sa' Sa0p&e*: 7oll the swab over the cleaned surface of a glass slide.
Pa%t >: Heat F/x/+
:eat fixing kills the bacteria in the smear firmly adheres the smear to the slide and allows the sample to more readily take up stains. •
0llow the smear to air dry.
•
0fter the smear has air4dried hold the slide at one end and pass the entire slide through the flame of a "unsen burner two to three times with the smear4side up.
Bow the smear is ready to be stained. ,lease Bote? Take care to prevent overheating the slide because proteins in the specimen can coagulate causing cellular morphology to appear distorted.
Pa%t ?: "%a0 Sta/+ P%o)e3%e
3. ,lace slide with heat fixed smear on staining tray. 5ently flood smear with crystal violet and let stand for 3 minute. -. Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle. D. 5ently flood the smear with 5ramHs iodine and let stand for 3 minute. @. Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle. The smear will appear as a purple circle on the slide. 6. /ecolori)e using G6 ethyl alcohol or acetone. Tilt the slide slightly and apply the alcohol drop by drop for 6 to 3 seconds until the alcohol runs almost clear. "e careful not to over4decolori)e. ;. $mmediately rinse with water. =. 5ently flood with safranin to counter4stain and let stand for @6 seconds. A. Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle. G. "lot dry the slide with bibulous paper. 3.
QUANTIFICATION OF MICROBES: SAMPLIN" AND SERIAL DILUTION BACTERIAL COUNT IN FOOD PRODUCTS TC AIM:
To estimate the total bacterial count in samples curd milk shake fruit 8uice sambar chutney soil tap water rotten tomato etc using spread plate techni%ue PRINCIPLE:
0s part of daily routine the laboratory microbiologist often has to determine the number of bacteria in a given sample as well as having to compare the amount of bacterial growth under various conditions. Enumeration of microorganisms is especially important in dairy microbiology food microbiology and water microbiology. There are many techni%ues for measuring microbial growth or population si)e but they can be divided into two main groups based on whether the population si)e is determined /%e)t&( or /+/%e)t&( . /irect counts include counting cells under the microscope *with or without special stains' using electronic particle counters or counting colonies on spread plates *also called a viable plate count'. $ndirect methods provide an estimate of cell numbers and can be done by measuring dry weight the optical density of a culture or by measurements of total protein. $ndirect methods have the advantage of being more rapid than direct methods but in order to be meaningful an indirect method must first be correlated to a direct method. A. T-e p&ate )o3+t <5/a'&e )o3+t#:
The number of bacteria in a given sample is usually too great to be counted directly. :owever if the sample is serially diluted and then plated out on an agar surface in such a manner that single isolated bacteria form visible isolated colonies the number of colonies can be used as a measure of the number of viable *living' cells in that known dilution. :owever keep in mind that if the organism normally forms multiple cell arrangements such as chains the colony4forming unit may consist of a chain of bacteria rather than a single bacterium. $n addition some of the bacteria may be clumped together. Therefore when doing the plate count techni%ue we generally say we are determining the number of &olony4(orming !nits *&(!s' in that known dilution. "y extrapolation this number can in turn be used to calculate the number of &(!s in the original sample.
Bormally the bacterial sample is diluted by factors of 3 and plated on agar. 0fter incubation the number of colonies on a dilution plate showing between D and D colonies is determined. 0 plate having D4D colonies is chosen because this range is considered statistically significant. $f there are less than D colonies on the plate small errors in dilution techni%ue or the
presence of a few contaminants will have a drastic effect on the final count. Likewise if there are more than D colonies on the plate there will be poor isolation and colonies will have grown together. 5enerally one wants to determine the number of &(!s per milliliter *ml' of sample. To find this the number of colonies *on a plate having D4D colonies' is multiplied by the number of times the original ml of bacteria was diluted *the dilution factor of the plate counted'. (or example if a plate containing a 3+3 dilution of the original ml of sample shows 36 colonies then 36 represents 3+3 the number of &(!s present in the original ml. Therefore the number of &(!s per ml in the original sample is found by multiplying 36 x 3 as shown in the formula below? The number of &(!s per ml of sample J The number of colonies *D4D plate' The dilution factor of the plate counted MATERIALS REQUIRED:
#terile nutrient agar plates sterile dilution tubes sterile 3 ml pipettes sterile tips for pipetteman sterile saline as a diluent glass spreader alcohol
PROCEDURE
3. Weigh 3 g of the sample in a sterile beaker and transfer to G ml of diluent in a conical flask. Mix well. This gives a 343 dilution. -. Transfer .6 ml of this diluted sample and mix with @.6 ml of sterile diluent in a test tube. D. #hake gently to facilitate mixing and dilution. @. ,repare serial dilution tubes by transferring @.6 ml of diluent into 6 sterile test tubes. 6. /ilute the supernatant serially to obtain34D 34@ 346 34; 34= 34A 34G343 respectively Transfer .3 ml of the appropriate dilution on the sterile B0 plates and spread them uniformly using alcohol sterili)ed cooled glass spreader. ;. $ncubate the plates at D=9& for -@ hours. =. &ount the number of colonies on the agar surface. A. &alculate the no. of bacteria present as cfu+ml + g of the given sample. G. /escribe the colony characteristics of the ma8or type of organisms seen on the plates used for counting.
RESULTS:
EP. No : Date : MICROBIOLO"ICAL QUALITY OF $ATER
The most important bacterial diseases transmitted by water are typhoid dysentery and cholera. #ince they are intestinal diseases causative agents are found in sewage. Therefore the presence of sewage in a water supply means that one or more of these disease4causing organisms may be present and that the water is potentially dangerous for human consumption. Co&/4o%0 o%a+/*0* /+ Seae
The coliform group is defined to include all aerobes facultative anaerobic gram4negative non4 spore forming rod4shaped species which ferment lactose with the production of acid and gas within @A h at D=9&. ,robably the most important members found in sewage polluted waters and relatively easy to isolate are E-coli, E. freundi and Aerobacter aerogenes.
#ome coliform species or varieties have been designated fecal because they are commonly found in fecesC others have been called non4fecal because they are believed to be normal inhabitants of soil. :owever in the tests which follow no attempt is made to differentiate between fecal and non fecal types. #uch a differentiation has been shown to be of limited value in determining the suitability of water for human consumption as contamination with either type renders the water potentially dangerous and unsafe from a sanitary standpoint.
M/)%oo%a+/*0* a* /+/)ato%* o4 ate% 93a&/t(
$n the routine microbiological examination of water to determine its potability it would not be satisfactory to base the test upon the presence of *or isolation of' pathogenic microorganisms for the following reasons?
3. ,athogens are likely to gain entrance into water sporadically but since they do not survive for long periods of time they could be missed in a sample submitted to the laboratory.
-. $f they are present in very small numbers pathogens are likely to escape detection by laboratory procedure.
D. $t takes -@ h or longer to obtain results from a laboratory examination. $f pathogens were present humans would be exposed to infection before actions could be taken to correct the situation.
I+/)ato% 0/)%oo%a+/*0*
The term indicator microorganismsN as used in water analysis refers to a kind of microorganism whose presence in water is evidence that the water is polluted with fecal material from humans or other warm4blooded animals. This kind of pollution means that the opportunity exists for the various pathogenic microorganisms which periodically occur in the intestinal tract to enter the water.
#ome of the important characteristics of an indicator organism are? 3. $t is present in polluted water and absent from unpolluted *potable' water. -. $t is present in water when pathogens are present. D. The %uantity of indicator organism correlates with the amount of pollution. @. $t has greater survival ability than pathogens. 6. $t has uniform and stable properties. ;. $t is harmless to humans and other animals. =. $t is present in greater numbers than pathog ens *making detection relatively easy'. A. $t is easily detected by simple laboratory techni%ues. #everal species or groups of bacteria have been evaluated for their suitability as indicator organisms. 0mong the organisms studied Escherichia coli and other coliform group bacteria most nearly fulfill the re%uirements of an ideal indicator organism and are regarded as the most reliable indicators of fecal pollution.
Escherichia coli a+ ot-e% )o&/4o%0 'a)te%/a
Escherichia coli is a normal inhabitant of the intestinal tract of humans and other warm4blooded animals. Bormally it is not pathogenic. 0nother member of the coliform group is Klebsiella pneumoniae which is widely distributed in nature. $t is found in soil water and grain and also in the intestinal tract of humans and other animals. Enterobacter aerogenes a coliform bacterium found in the intestinal tract of humans and other animals occurs also in soil water and dairy products.
The coliforms as a group are characteri)ed as gram4negative non4spore forming aerobic and facultatively anaerobic rod4shaped bacteria that ferment lactose with the production of acid and gas within @A h at D6 P&.
The coliforms have several characteristics in common with members of the genera Salmonella and Shigella two genera which are enteric pathogenic species. :owever a ma8or distinctive biochemical difference is that the )o&/4o%0* 4e%0e+t &a)to*e /t- p%o3)t/o+ o4 a)/ a+ a*
Salmonella a+ Shigella o +ot 4e%0e+t &a)to*e. The fermentation of lactose is the key reaction in the laboratory procedure performed to determine potability of water.
Sa0p&/+ o4 ate%
(or collection of sample great care is necessary. The water samples collected for bacteriological analysis should ensure truly representative samples from different sources and prevent extraneous contamination during collection.
P%o)e3%e*
&ollect the sample in sterili)ed ground glass stoppered bottle of about D46 ml capacity. While collecting from top allow the water to run for D4@ m. #terili)e the no))le of the top by heating it with a burner or with a piece of cotton wool which is dipped in spirit. 0gain allow the water to flow slowly for a minute and then holding the sample bottle in one hand remove stopper with other hand. (lame the mouth %uickly and allow the bottle to fill. 7eplace the stopper.
Most ,robable Bumber *M,B' Estimates These are based on assumption that bacteria are QnormallyH distributed in li%uid media that is repeated samples of the same si)e from one source are expected to contain the same number of organisms on average. #ome samples will obviously contain a few more some a few less. T-e a5e%ae +30'e% /* t-e 0o*t p%o'a'&e +30'e% . This techni%ue is used mainly for estimating coliforms but it can be used almost for any organism in li%uid samples if growth can be easily observed e.g. by turbidity or acid production. Examples are yeasts and molds in fruit 8uices and beverages &lostridia in food emulsions. (or anaerobes back tube M,B counts can also be done. /ouble strength broth is used for the larger volumes because the medium would otherwise be too dilute.
$t is possible to calculate the most probable number of organisms per 3ml for any combination of results from such sample series. Tables have been prepared for samples of 3ml 3ml and .3 ml using five tubes or three tubes of each sample si)e. Tables indicate the estimated no. of bacteria of the coliform group present in 3 ml of water corresponding to various combinations of positive and negative results in the amounts used for the tests. The tables were basically computed by Mc&ready and therefore are referred to as Mc&readyHs table.
Procedure 3. $noculate 3 ml of water sample into each of D Lauryl Tryptose *LT' broth tubes *double strength'. -. $noculate 3ml and .3 ml of water sample into each of D LT broth tubes *single strength'. D. $ncubate all tubes at D=P& for -@ to @A h.
@. 0ny amount of gas in the inverted /urhamHs tube constitutes a positive test. The sample must be collected in a sterile bottle. The sample must be representative of the supply from which it is taken. &ontamination of the sample must be avoided during and after sampling. The sample should be tested as promptly as possible after collection. $f there is a delay in examination of the sample it should be stored at a temperature between and 3P&. The routine bacteriological procedure consists of *3' 0 plate count to determine the number of bacteria present and *-' Tests to reveal the presence of coliform bacteria.
Sta+a% p&ate )o3+t
&olony counts are performed after plating samples of the water. ,late4count standards have not been suggested for water because water with a few pathogenic bacteria is obviously more dangerous than water containing many saprophytic bacteria. Bevertheless water of good %uality is expected to give a low total count less than 3 per milliliter. ,late counts are useful in determining the efficiency of the operations removing or destroying organisms4sedimentation filtration and chlorination. 0 count can be made before and after the specific treatment. The results indicate the extent to which the microbial popu lation has been reduced. Te*t* 4o% t-e ete)t/o+ o4 )o&/4o%0 'a)te%/a
#everal selective and differential media greatly expedite the examination of water for coliform organisms. The examination involves three successive steps? *3' ,resumptive test *-' &onfirmed test and *D' &ompleted test
Multiple tube fermentation techni%ue is followed here. The routine standard tests are *0' ,resumptive *"' &onfirmed *&' &ompleted test E,E7$MEBT A/0
To determine whether the given water sample is potable Re93/%e0e+t*
Mc&onkey broth EM" or Endo agar plates "rilliant green lactose bile broth *"5L"' with inverted /urhamHs tube B0 ,lates and water sample. P%o)e3%e: (A) Presumptive Test
*3' $noculate 6ml of water sample in 6 test tubes-.6ml in 6 test tubes3ml in 6 test tubes containing 6 ml of Mcconkey broth . *-' $ncubate all tubes at D=P& for -@4@A h. 0ny amount of gas in the inverted /urhamHs tube constitutes a positive presumptive test. The absence of gas formation within that period constitutes negative test and no further tests need to be performed. (B) Confirmed Test
(rom tubes showing positive presumptive test inoculate a loopful into "5L" and streak a loopful on EM" or Endo agar incubate the tubes and the plates at D=P& for @Ah. 5as in the "5L" tubes or typical colonies on EM" or Endo agar4 dark centered pink colonies on these media constitutes positive confirmed.
(C) Completed Test
3.
,ick up one typical coliform colony from EM" or Endo agar plate and subculture it on a B0 slant.
-.
,repare a suspension from each colony and inoculate a loopful into LT"
D.
$ncubate the slant and broth tube at D=P& for -@ h and observe for gas in the LT" tube
Eo*/+ 0et-(&e+e '&3e aa%
This medium is prepared by adding definite %uantities of the two stains eosin and methylene blue to a melted lactose agar base. 0 loop4full of culture from each positive fermentation tube is streaked over the surface of EM" agar. The plates are inverted and incubated at D=P& for -@ h. $t is used for the isolation cultivation and differentiation of 5ram4negative enteric bacteria based on lactose fermentation. "acteria that ferment lactose especially the coliform bacterium Escherichia coli appear as colonies with a green metallic sheen or blue4black to brown color. "acteria that do not ferment lactose appear as colourless or transparent light purple colonies. &olonies of Yersinia pseudotuberculosis are pale pink.
Three types of colonies develop on the medium. 3.
Typical 4 nucleated with or without metallic sheen
-.
0typical 4 Fpa%ue non4nucleated pink
D.
Begative 4 0ll others.
$f typical coliform colonies appear on the plates the confirmed test may be considered positive. $f only atypical colonies appear the confirmed test cannot be considered negative since some coliforms fail to produce typical colonies on this medium or the colonies develop slowly. $f no colonies or non4coliforms colonies develop within -@ h the confirmed test may be considered negative.
The colour of coliform on this medium depends on - factors *3' the reaction of eosin *an acid stain' with methylene blue *a basic stain' to form a c ompound of either acidic or neutral in nature and *-' the formation by lactose4fermenting organisms of sufficient acid to cause this stain compound to be taken up by individual cells of a colony. The non4lactose4fermenting organisms are not coloured because the stain compound is not taken up in basic solution.
E+o aa% Metallic gold4like sheen imparted to the surface of the typical colonies. The media is used for the selective isolation cultivation and differentiation of coliform and other enteric microorganisms based on their ability to ferment lactose. Lactose fermenting bacteria appear as dark red colonies with a gold metallic sheen. Lactose4non4fermenting bacteria appear as colourless or translucent colonies.
B%/&&/a+t %ee+ &a)to*e '/&e '%ot 0 positive test is indicated by the presence of gas in any amount in the inverted vial within
incubation period.
RESULT:
Exp : Date: MICROBIOLO"ICAL QUALITY OF MIL AIM:
To evaluate the microbiological %uality of milk. INTRODUCTION:
Milk is one of the most important foods for man but it is also highly susceptible to microbial contamination and spoilage. 0 variety of microorganisms including several pathogenic species can gain entry into milk during itHs production and handling. 0 knowledge of the numbers and types of bacteria present in raw milk supplies is thus very useful in determining the hygienic conditions of itHs production and handling itsH keeping %uality and itHs suitability for processing or manufacture of products. 0 number of bacteriological tests are used for checking the %uality of milk and these may be broadly grouped into *i'
/irect enumeration of total bacterial population in milk *e.g. direct microscopic count'
*ii'
Estimation of the numbers of viable bacteria e.g. standard plate count.
*iii' Testing for the presence of specific types of contaminants *e.g. coliform test' *iv' 0ssessing the metabolic activities of bacteria *e.g. methylene blue and resa)urin reduction tests' *v'
Estimating the chemical changes or products formed in milk by bacterial growth *e.g. acidity gas production p: and proteolysis'
When %uality of milk has to be detected on the spot it is necessary to adopt simple reliable and rapid methods of bacteriological examination. Two such methods are usually followed.
Met-(&e+e '&3e %e3)t/o+ te*t I+t%o3)t/o+
This test is based on the principle that methylene bluet *an oxidation4reduction dye or indicator' which is blue in its oxidised state is reduced to a colourless compound *Leuco form' as a result of the metabolic activities of bacteria in milk. When a solution of methylene blue is added the organism present in milk consume the dissolved oxygen and lower the F47 potential to a level when methylene blue and similar indicators are reduced or decolourised. The time taken for the reduction of the dye *methylene blue reduction time' is influenced by the number and types of bacteria growing in milk. The greater the number of organisms present in milk and greater their activity the more rapidly is the dye reduced. The methylene blue reduction time thus gives an indication of bacterial numbers and activity in milk. The M.".7. test is therefore used for *i' 8udging the hygienic %uality of milk and grading raw milk supplies *ii' for assessing the probable %uality of milk and *iii' for detecting post pasteurisation contamination in milk.
Mate%/a&*
3.
Thermostatically controlled water bath maintained at D=P&.
-.
#terile test tubes without rim *36 x 3; mm' preferably with marking at 3 ml.
D.
#terili)ed rubber bungs to fit into the above test tubes. The rubber bungs together with forceps are held in boiling water for 3 minutes prior to use.
@.
3. ml and 3. ml pipettes.
6.
&lock watch or an interval times
;.
(orceps beakers and flasks.
=.
#tandard methylene blue solution.
A.
(our samples of milk in sample bottles * fresh raw milk raw mik refrigerated raw milk refrigerated after - hours pasteuri)ed milk'
Met-(&e+e B&3e So&3t/o+ 0 standard solution of methylene blue is prepared by dissolving one tablet of approved methylene blue thiocyanate of chloride in - ml of cold sterile glass distilled water in a sterile flask by gentle heating in water bath or by allowing the mixture to stand for several hours to facilitate complete solution and then adding ; ml of sterile glass distilled water. Fne ml of this solution mixed with 3 ml of milk results in obtaining a final concentration of 3+D for the dye which has been found to be satisfactory for the test. The stock solution must be stored in a sterile glass4stoppered amber coloured bottle in a dark place. (resh solution must be prepared once in two months. P%o)e3%e
3.
Thoroughly mix the sample of the milk.
-.
Transfer 3 ml of each sample of milk into a test tube.
D. 0dd 3 ml of the methylene blue solution to the milk in the test tubes and replace the cotton plugs with sterile rubber bungs using sterile forceps. While transferring methylene blue solution care should be taken not to contaminate the pipette by touching the milk or otherwise a fresh pipette will have to be used for transferring methylene blue solution to another tube.
@. Mix the dye and the milk by inverting the tubes twice.
6.
,lace the tubes in the water bath.
;.
Fbserve the test tubes after every D minutes and if there is no sign of reduction *decolourisation' the tubes are inverted once and returned to the water bath. $f the decolourisation has commenced the tubes should not be inverted or shaken.
=.
G. 3.
&ontinue the observation until the complete reduction of the dye *complete decolourisation' occurs or the formation of a persistent blue ring *.6 mm' at the top. A. Two control tubes one containing 3 ml of milk and 3 ml of the methylene blue solution after heating it in boiling water for D minutes and another with 3 ml of milk plus 3 ml of tap water are also kept in the water4bath. These are re%uired for comparing the colour changes in experimental tubes 7ecord the times taken for reduction of methylene blue
Tabulate the results
I+te%p%etat/o+
The following standard for methylene blue reduction times are suggested as a guide for grading of raw milk supplies. M.B.R. t/0e
Q3a&/t( o4 0/&2
6 and above
D and @
5ood
3 and -
(air
3+- and below
,oor
RESULT:
EP: DATE:
ENUMERATION OF LACTIC ACID BACTERIA FROM FERMENTED FOODS ! PLATE COUNT METHOD AIM
To estimate the total bacterial count *Lactic acid bacteria' in samples of curd soy sauce yakult batter butter milk Fld rice using pour plate techni%ue PRINCIPLE
0s part of daily routine the laboratory microbiologist often has to determine the number of bacteria in a given sample as well as having to compare the amount of bacterial growth under various conditions. Enumeration of microorganisms is especially important in dairy microbiology food microbiology and water microbiology. There are many techni%ues for measuring microbial growth or population si)e but they can be divided into two main groups based on whether the population si)e is determined /%e)t&( or /+/%e)t&( . /irect counts include counting cells under the microscope *with or without special stains' using electronic particle counters or counting colonies on spread plates *also called a viable plate count'. $ndirect methods provide an estimate of cell numbers and can be done by measuring dry weight the optical density of a culture or by measurements of total protein. $ndirect methods have the advantage of being more rapid than direct methods but in order to be meaningful an indirect method must first be correlated to a direct method. A. T-e p&ate )o3+t <5/a'&e )o3+t#:
The number of bacteria in a given sample is usually too great to be counted directly. :owever if the sample is serially diluted and then plated out on an agar surface in such a manner that single isolated bacteria form visible isolated colonies the number of colonies can be used as a measure of the number of viable *living' cells in that known dilution. :owever keep in mind that if the organism normally forms multiple cell arrangements such as chains the colony4forming unit may consist of a chain of bacteria rather than a single bacterium. $n addition some of the bacteria may be clumped together. Therefore when doing the plate count techni%ue we generally say we are determining the number of &olony4(orming !nits *&(!s' in that known dilution. "y extrapolation this number can in turn be used to calculate the number of &(!s in the original sample.
Bormally the bacterial sample is diluted by factors of 3 and plated on agar. 0fter incubation the number of colonies on a dilution plate showing between D and D colonies is determined. 0 plate having D4D colonies is chosen because this range is considered statistically significant. $f there are less than D colonies on the plate small errors in dilution techni%ue or the presence of a few contaminants will have a drastic effect on the final count. Likewise if there are
more than D colonies on the plate there will be poor isolation and colonies will have grown together. 5enerally one wants to determine the number of &(!s per milliliter *ml' of sample. To find this the number of colonies *on a plate having D4D colonies' is multiplied by the number of times the original ml of bacteria was diluted *the dilution factor of the plate counted'. (or example if a plate containing a 3+3 dilution of the original ml of sample shows 36 colonies then 36 represents 3+3 the number of &(!s present in the original ml. Therefore the number of &(!s per ml in the original sample is found by multiplying 36 x 3 as shown in the formula below? The number of &(!s per ml of sample J The number of colonies *D4D plate' The dilution factor of the plate counted MATERIALS REQUIRED:
#treile nutrient agar plates sterile dilution tubes sterile 3 ml pipettes sterile tips for pipetteman sterile saline as a diluent glass spreader alcohol
PROCEDURE
3. Weigh 3 g of the sample in a sterile beaker and transfer to G ml of diluent in a conical flask. Mix well. This gives a 343 dilution. -. Transfer .6 ml of this diluted sample and mix with @.6 ml of sterile diluent in a test tube. D. #hake gently to facilitate mixing and dilution. @. ,repare serial dilution tubes by transferring @.6 ml of diluent into 6 sterile test tubes. 6. /ilute the supernatant serially to obtain34D 34@ 346 34; 34= respectively. ;. Transfer .3 ml of the appropriate dilution on the sterile petri plates and spread uniformly using alcohol sterili)ed cooled glass spreader. =. $ncubate the plates at D=9& for -@ hours. A. &ount the number of colonies on the agar surface. G. &alculate the no. of bacteria present as cfu+ml + g of the given sample. 3. /escribe the colony characteristics of the ma8or type of organisms seen on the plates used for counting. RESULTS:
Exp. No: Date: ENUMERATION OF YEAST AND MOLDS AIM:
To enumerate the yeast and molds from different food. MATERIALS REQUIRED:
(ood samples*tomato potato carrot custard apple 8uice guava 8uice cocnut chutney' sterile plates with potato dextrose agar containing 3mg+ml of chloramphenicol sterile test tube with @.6ml .3 peptone micropipette sterile beakers sterile tips sterile spatula and spreader rod.
PROCEDURE:
•
Weigh .6g of sample or .6 ml of li%uid sample using a sterile beaker or sterile pipette. Take @.6 ml of peptone water *.3' and add the samples to it. ,repare serial dilution using = test tubes containing .3 peptone water to obtain 34D
•
34@ till 34= !sing pour plate techni%ue transfer 3 ml of appropriate dilution on sterile petri plates
•
and pour the potato dextrose agar solution into the petri plates and allow it to solidify $ncubate the plates*--P& to -6P& ' for 6 days and count the plates containing 36 to 66
• •
colonies and record it chloramphenicol or gentamycin are used because
RESULT:
Exp. No: Date: ENUMERATION OF SPORES FROM PEPPER AIM:
To enumerate the mesophilic bacterial spores from species. PRINCIPLE:
"acteria produce spores in response to environmental stress. #pores are dominant forms of cells which are essential to heat dehydration free)ing and irradiation compared to vegetative forms. #pores forming bacteria are Clostridium and Bacillus. Members of the former are strict or
facultative anaerobes whereas the latter are aerobes of facultative anaerobes. They are both mesophilic spore forming bacteria. . PROCEDURE:
3.
Weigh 3 g of sample in a sterile beaker and add it to GG ml of sterile .3 peptone water.
-.
Mix well put the samples in a water bath at A9 & for D m.
D.
This will heat shock the spores and kill the vegetative bacteria.
@.
7emove the flask from water bath mix well and allow the particles to settle down.
6.
Transfer .6 ml of supernatant to a test tube containing @.6 ml of .3 sterile peptone water. Mix well this gives 3 R D dilution.
;.
#imilarily prepare 3 R @ 3 R 6 dilutions.
=.
,late each dilution in %uadruplicate using the pour plate techni%ue and molten nutrient agar at @69 &.
A.
$ncubate teh plates at D69 & and 69 &.
G.
0t the end of -@ h count the colonies and report mesophilic aerobic spore count and thermophilic aerobic spore count
RESULT:
Exp. No: Date: INHIBITORY EFFECT OF SPICES ON MICROBIAL LOAD IN RA$ POULTRY AIM:
To evaluate the inhibitory effect of spices on microbial load in raw poultry. MATERIALS REQUIRED:
5rounded spice sample*5arlic 5inger pepper turmeric' fleshy chicken sterile plates with nutrient agar sterile distilled water peptone water *.3' micropipette sterile beakers sterile tips sterile spatula and spreader rod. PROCEDURE: 1. Weigh 1 gm of sample using sterile spatula into a sterile container(test tube). 2. Add 9ml of 0.1% sterile peptone water . 3. ransfer 300>l of sample from the test tube on sterile !A plates and spread them uniforml" using alcohol sterili#ed$ cooled glass spreader. . Add 2 gm of sample to & ml of distilled water in a sterile test tube to prepare 20 % concentration (dilution). '. hen add ' ml of the 20 % solution to ' ml of distilled water to prepare 10% dilution in another test tube. . hen add ' ml of the 10 % solution to ' ml of distilled water to prepare ' % dilution in another test tube. . *" well di+usion method punch a small holes using the sterile tips in the agar plates &. ransfer 200>l of the prepared dilution in three di+erent petri plates e,actl" in the small holes made in well di+usion process. 9. -epeat the same step for all the samples . 10.noculate the plates at 3 P/ for & hrs . 11.Absorb the plates and tabulate the results .
RESULT:
Exp.No Date ENUMERATION AND ISOLATION OF E.COLI FROM PROCESSED MEAT CHICEN AIM:
To enumerate E.&oli which may be present in foods. :omogeni)e with Gml of sterile peptone REQUIREMENTS:
(ood samples *7aw meatchicken fish'EM" peptone*.3'sterile piptte tips stirrer. PROCEDURE:
agar flask
containing G ml sterile
3. Weight 3g of sample using a sterile spatula into a sterile container*test tube'. -. 0dd G ml of peptone water*this gives 3?3 dilution'. D. Transfer 3 ml of sample from the test tubes on sterile petri plates and pour EM" agar and allow it to slodify*pour plate techni%ue'. @. 7epeat the same procedure for all the D samples . 6. $noculate the plates at D=P& for -@ hrs. ;. Fbserve the plates and count the colonies on the media. RESULT:
Exp. No: Date: THERMAL DESTRUCTION OF MICROOR"ANISMS : TDT AND TDP
AIM
To study the effect of high temperature a physical method on the destruction of microbes in li%uid suspension. PRINCIPLE:
:eat is the most used method for inactivating the microorganisms in food production. Microbial exposure to heat has two parameters4temperature and exposure time. :eat appears to kill microorganisms by denaturing their en)ymes. :eat resistance varies among different
microbes. These differences can be expressed through the concept of thermal death point. T-e%0a& eat- po/+t
#terile suspension tubes sterile 3 ml pipettes sterile recovery tubes containing sterile nutrient broth sterile saline thermometers and water bath. DETERMINATION OF TDP:
3. To @.6 ml of .3 sterile peptone water add .6 of 8uice sample this will give 3?3 dilution -. #imilarly prepare 34- and 34D respectively. D. To prepare 34@ dilution to @6 ml .3 sterile peptone water add 6ml of sample from 3 4D dilution in a sterile conical flask. @. Transfer D ml of sample from 34@dilution to D sterile test tube. 6. 0d8ust and fix the temperature of water bath to =Pc APc and GPc respectively for 3 minutes. ;. &ool the test tubes =. Then Transfer D> of the sample from the test tubes on the sterile B0 plates and spread them uniformly using alcohol sterili)ed cool glass spreader. A. $ncubate the plates at D=P& for -@ hrs count the no. of colonies on the agar surface and tabulate the results and to determine T/, DETERMINATION OF TDT:
3. (rom the 34@ dilution of sample prepared above transfer D ml of sample to 3- sterile test tubes. -. 0d8ust the temperature of the water bath to =P& AP& GP&. D. $ncubate @ test tubes in each water bath kept at three different temperatures.
@. 7emove one tube at a regular interval of 6 3 36 and - mins respectively from each water bath. 6. &ool the test tubes. ;. Then transfer D>l of sample from the test tubes on the sterile B0 plates and spread them uniformly using sterili)ed cooled glass spreader. =. $ncubate the plates at D=P& for -@ hrs A. Tabulate the results and count the no of colonies and determine the T/T. RESULT:
EP .NO: DATE: EFFECT OF CLEANIN" AND DISINFECTION ! PHENOL COEFFICIENT TEST AIM:
To determine the effectiveness of some chemical disinfectants used as antimicrobial agents and calculate its phenol coefficient. PRINCIPLE: Microorganisms are present everywhere and one must be constantly aware of the living
invisible world. There is a strong need to kill bacteria when and where their presence is undesirable. Therefore many situations such as preparation
of surgical operations
microbiological studies and disinfection of infectious materials call forth the need and use of methods to destroy them. The destruction of microorganisms may be achieved by physical and chemical means. #terili)ation is defined as the process where all the living microorganisms including bacterial spores are killed. /isinfection is the process of elimination of most pathogenic microorganisms *excluding bacterial spores' on inanimate ob8ects. #terili)ation is always aimed at both pathogenic and nonpathogenic bacteria while disinfection in its true sense applies only to the pathogenic ones so that there is no or a much reduced threat of disease. 0 disinfectant is any agent such as heat or a chemical *like iodine' that kills pathogenic microorganisms. D/*/+4e)ta+t* are used to reduce the numbers of microbes on non4living surfaces while a+t/*ept/)* are used to reduce the microbial population on living tissue. 0ntiseptics normally are more bacteriostatic in that they prevent bacterial multiplication but do not kill the organism. The emergence of anti4microbial soaps lotions and other products has seen a huge increase over the last few years. The number of choices is excessive and the consumer is often unaware that many of the anti4microbial agents are no more effective than basic soap and water. The effectiveness of an anti4microbial is dependent upon many factors such as the concentration of the antimicrobial agent the amount of contamination the sensitivity of the contaminating organisms temperature and length of exposure. This exercise evaluates the influence that specific antimicrobial agents may or may not have on bacterial growth.
Many factors influence the effectiveness of chemical disinfectants and antiseptics. The 0/)%o'/)/a& *to kill' or 0/)%o'/o*tat/) *to inhibit' e44/)/e+)( of a chemical is often determined with respect to its ability to deter microbial growth. More specifically the microbicidal efficiency of a chemical is often determined with respect to phenol and is known as the p-e+o& )oe44/)/e+t
0 list of commonly used antiseptics and disinfectants and their area of application is shown in table.
Ae+t* U*e to Co+t%o& M/)%o'/a& "%otA. D/*/+4e)ta+t* a+ A+t/*ept/)*
3. ,henols and phenolics 4 these compounds inactivate proteins denature en)ymes and in8ure plasma membranes and should only be used on surfaces. Examples include Lysol hexachlorophene and p:iso:ex. -. :alogens R may be used on surfaces either alone or as components of organic or inorganic solutions to inactivate en)ymes and other cellular proteins. Tend to be strong oxidi)ing agents. $odine combines with the amino acid tyrosine chlorine when added to water forms hypochlorous acid. "etadine is another example often used instead of iodine. D. 0lcohols R denature proteins and dissolve lipids. Examples include ethanol and isopropanol. @. :eavy metals R such as silver mercury copper and )inc exert their influence through oligo4 dynamic action such as combining with the sulfhydryl *4#:' groups and denaturing proteins. Examples include silver nitrate mercurochrome and copper sulfate. 6. #urface active agents R soaps and detergents decrease the tension between molecules that lie on the surface of a li%uid ;. Suaternary ammonium compounds *%uats' Rcationic detergents attached to B:@ disrupt plasma membranes denature proteins and inhibit en)ymes. Examples include &epacol and Uephran. =. Frganic acids R used in the food and cosmetic industry to prevent growth of microorganisms. Examples include sorbic acid ben)oic acid and propionic acid. A. 0ldehydes R formaldehyde and glutaraldehyde attach methyl or ethyl groups to /B0 and proteins making them nonfunctional. B. A+t/'/ot/)*
3. $nhibition of cell wall synthesis ! may inhibit synthesis of petidogylcan. $nclude penicillins cephalosporins vancomycin bacitracin oxacillin and nafcillin -. /amage to plasma membrane R polymyxin " nystatin and amphotericin " D. $nhibition of protein synthesis R streptomycin *causes misreading of codons on m7B0' chloramphenicol *prevents peptide bond formation between amino acids' tetracyclines *prevents
hydrogen bonding between anticodon on t7B04aa complex and codon on m7B0' kanamycin erythromycin and gentamicin @. $nhibition of nucleic acid synthesis R rifamycin actinomycin / nalidxic acid ciprofloxacin and norflaxacin 6. #tructural analogs R such as sulfonamides that are structurally similar to cellular metabolites and compete with these in en)ymatic reaction.
MATERIALS REQUIRED:
#terile nutrient broth tubes -@ hour culture of E.coli phenol commercial disinfectants such as Lysol /ettol test4tube rack "unsen burner inoculating loop alcohol. The phenol is diluted with tap water to obtain 3?A 3?G and 3?@ dilutions. The dettol and ly)ol sodium hypochlorite are diluted with tap water to obtain 3?@ 3?@6 and 3?6 dilutions. PROCEDURE:
3. Label a set of G nutrient broth test tubes for 3 disinfectants for D different dilutions with name and dilution of disinfectant and time interval of sub4culturing . -. ,lace the test tubes with disinfectant dilutions in separate sacs. D. !sing pipette rapidly introduced .6 ml *3drop' of the E.coli culture into the test tubes with disinfectants. note the time of inoculation. @. Mix the tubes well to ensure contact of the disinfectant with microbe . 6. 0t intervals of 6 3 and 36 mins using sterile techni%ue transfer one loop full from each test tube into the appropriate sterile tube of nutrient broth . ;. $noculate all cultures for the presence of growth . =. 0bsorb all cultures for the presence of growth. A. 7ecord for the presence of growth and for absence of growth . G. Tabulate the results for three disinfectants . 3. 7epeat for all three disinfectants.
Re*3&t:
