313 PHT

Lab. No. 8

Gram’s Stain

Gram’s +ve Gram’s -ve

Cocci Bacilli Cocci Bacilli

AerobicAerobic, , non-fermentativenon-fermentative, , motilemotile, , oxidase-positiveoxidase-positive gram-negative bacilli.gram-negative bacilli.

Most Important SpeciesMost Important SpeciesP.aeruginosa P.aeruginosa

opportunistic pathogen opportunistic pathogen causes UTI, wound infections and causes UTI, wound infections and

otitis mediaotitis media

Pseudomonas

Microscopical examination:Microscopical examination:

(morphology)(morphology)

A) Gram’s Stain:A) Gram’s Stain:

Gram –ve Gram –ve

Non-sporeforming bacilli , Non-sporeforming bacilli ,

having single arrangementhaving single arrangement..

B) Examination of Motility:B) Examination of Motility:

Using the Using the “Hanging Drop technique”“Hanging Drop technique”

Pseudomonas is highly motile by means of Pseudomonas is highly motile by means of polar flagella.polar flagella.

Cultural Cultural characteristic:characteristic:

It grows on It grows on simple media.simple media.

It usually produces It usually produces exopigments.exopigments.

1) Growth on nutrient 1) Growth on nutrient agar:agar:

Its growth on nutrient agar Its growth on nutrient agar

showing showing greenish greenish discolourationdiscolouration

due to due to exopigmentexopigment production. production.

Cetrimide agar is a Cetrimide agar is a highly selective highly selective mediummedium for pseudomonas species due for pseudomonas species due to presences of to presences of cetrimide cetrimide which which inhibits the growth of other bacteria.inhibits the growth of other bacteria.

It contains also It contains also MgClMgCl22 & & KK22SoSo44 to to facilitate production of the facilitate production of the charactaristic charactaristic green pigment green pigment of of pseudomonas. pseudomonas.

2) Growth on Cetrimide Agar:2) Growth on Cetrimide Agar:

Principle:

Results:

Only Pseudomonas species can grow on cetrimide agar showing growth of pale colonies with diffusion of green pigmentation.

MacConkey’s agar is a selective and differential medium

selective medium for selective medium for enteric enteric gram –ve gram –ve bacteriabacteria ( (bile saltbile salt inhibit the growth of inhibit the growth of non enteric bacteria).non enteric bacteria).

Test sugar:Test sugar: lactose.lactose. pH indicator:pH indicator: neutral neutral

redred ( yellow in alkaline, ( yellow in alkaline, pink in acidic pH).pink in acidic pH).

3) Growth on MacConkey’s agar:3) Growth on MacConkey’s agar:

Principle:

Biochemical reaction:Biochemical reaction:

1)Oxidase test.1)Oxidase test.

2) Nitrate Test.2) Nitrate Test.

3) Oxidation Fermentation (O/F) Test.3) Oxidation Fermentation (O/F) Test.4) Growth on Triple Sugar Iron (TSI)

agar.

Results:

+ve Test: Appearance of purple colour within few seconds.

purple colour

+ve testPseudomonas

No colour

-ve testEnterobacteriaceae

1)Oxidase test:1)Oxidase test:

2) Nitrate Test:2) Nitrate Test:

Principle:

NitrateNitrate reductase

nitrite

α-naphthyl amine (nit. A)

Sulphanilic acid (nit. B)

Red diazonium saltEnterobacteriaceaeEnterobacteriaceae

Further reduction

Nirtogen (N2)

Add zinc dust (reducing agent)

If no red colour!

Procedure:

Nitrate broth

test m.o

Nit.A

Nit. B

Red colour

No red colour

Add zinc dust

Incubate at 35oC for 24

hrs

Results:

Red colour after addition of nit.A & nit.B

Reduction of Nitrate to nitrite

EnterobacteriaceaEnterobacteriaceaee

Red colour after addition of zinc dust

-ve reduction

No red colour after addition of zinc dust

Further reduction to

NitrogenPseudomonas

3) Oxidation Fermentation 3) Oxidation Fermentation (O/F) Test:(O/F) Test:

Sensitive O/F testSensitive O/F test

Positive Test:

O-/F- O+/F+ O+/F- O-/F+

Results:

Fermentative

EnterobacteriaceEnterobacteriaceaeae

Oxidative

Pseudomonas

Non

Saccharolytic

Principle:

butt slant

4) Growth on Triple Sugar Iron(TSI) agar:

Results:

1. No Fermentation:1. No Fermentation:

Butt: alkaline (red) Slant: alkaline (red)

Results:

Butt:

Slant:

H2S :

acidic (yellow)

acidic (yellow)

-ve

acidic (yellow)

alkaline (red)

-ve

acidic (yellow)

alkaline (red)

+ve

alkaline (red)

alkaline (red)

-ve

Pseudomonas aeruginosa

I'm also veryI'm also very resistant to resistant to

most most antibioticsantibiotics, , so it's very so it's very hard to get hard to get

rid me. rid me.

Water

Milk

Air

Examination of water, milk Examination of water, milk and airand air

Importance of water Importance of water examination for pathogens examination for pathogens

Water intended for human Water intended for human

consumption should not contain consumption should not contain

any pathogenic organisms.any pathogenic organisms.

Water is used for many Water is used for many

applications either at home for applications either at home for

cooking ,washing or drinking or cooking ,washing or drinking or

in industries such as food and in industries such as food and

pharmaceuticals.pharmaceuticals.

It is also important for It is also important for hospitals for example hospitals for example haemodialysis unithaemodialysis unit

Testing of water samples Testing of water samples are done regularly to are done regularly to make sure of its safetymake sure of its safety

Supplies of drinking water Supplies of drinking water contaminated with sewage may contaminated with sewage may cause diseases such as: cause diseases such as: typhoid fever and cholera.typhoid fever and cholera.

All sources of water should be All sources of water should be tested regularly. tested regularly.

Microorganisms which indicate Microorganisms which indicate the fecal pollution in water are the fecal pollution in water are usually common intestinal usually common intestinal commensal bacteria. commensal bacteria.

Most important indicators of Most important indicators of fecal pollution of waterfecal pollution of water

Escherichia coli:Escherichia coli: The essential indicator of fecal pollution of human /animal origin.The essential indicator of fecal pollution of human /animal origin. It is an important member of the coliform bacteria.It is an important member of the coliform bacteria.

Coliforms are members of the enterobacteriaceae family and theyColiforms are members of the enterobacteriaceae family and they1.1. grow in the presence of bile salts.grow in the presence of bile salts.

2.2. produce acid and gas from fermentation of lactose at 37°C.produce acid and gas from fermentation of lactose at 37°C.

It is the commonly-used bacterial indicator of sanitary quality of food It is the commonly-used bacterial indicator of sanitary quality of food and water.and water.

Enterococcus faecalis:Enterococcus faecalis:• less numerous than E.coli in less numerous than E.coli in

human feces, but more resistant to human feces, but more resistant to chlorination.chlorination.

Clostridium perfringens:Clostridium perfringens:• Less numerous in human fecesLess numerous in human feces• Its spores can survive in the Its spores can survive in the

environment environment • Resist treatment processes than Resist treatment processes than

most of the indicators. most of the indicators.

Media used in bacteriological examination of water

1. For coliforms: MacConkey’s broth Containing bromocresol purple as the pH indicator.

To confirm the presence of E.coli : EMB agar + IMVC

Enterococcus faecalis:Enterococcus faecalis: Glucose azide broth.Glucose azide broth.

Clostridium perfringes:Clostridium perfringes: Differential reinforced clostridial medium.Differential reinforced clostridial medium.

Membrane Filtration MethodMembrane Filtration Method

Determination of Most Probable Number Determination of Most Probable Number (MPN) by dilution method (MPN) by dilution method

Pour plate techniquePour plate technique

Methods Used in Bacteriological Examination

of Water

• Using Millipore Filter Apparatus

Membrane Filtration Method

MacConkey’s agar

Determination of MPN of Coliforms by Dilution Method

Water Sample

50 mlDSMB

5 x 10 mlDSMB

5 x 5 mlSSMB

50 ml watersample 10 ml water

sample1 ml water

sample

Results:Results:

Positive tubes:Positive tubes: showing production of showing production of acid or gas.acid or gas.

Acid production:Acid production: change color of tube change color of tube from purple to yellowfrom purple to yellow

Gas production:Gas production: detected in the detected in the Durham’s tube.Durham’s tube.

Purple Yellow

Gas

Determine no. of coliforms per 100 ml water sample (MPN) using the standard probability

table.

1 3 2

MPN = 14

i.e: No. of coliform bacilli per 100 ml water sample is 14 cells.

Most probable number of Most probable number of coliforms by McCrady’s tablecoliforms by McCrady’s table

Using 10 fold serial dilution method

Viable Bacterial Count

9 ml Saline1 2 3Water sample

1 ml water

1 ml 1 ml

1/101/10 x 1/10

1/1001/100 x 1/10

1/1000

1 ml 1 ml 1 mlMelted NA

1 2 3

Results:Results:

DilutioDilution n

factorfactor 11 22 33 XXX . yX . y

1010 xx11 X1.y1

101022 xx22 X2.y2

101033

xx33 X3.y3

No. of colonies per plateY

No. of cells per 1 ml = X1.y1 + X2.y2 + X3.y3

3

Water

Milk

Air

Examination of water, milk Examination of water, milk and airand air

Human infections may be caused byHuman infections may be caused by

theingestion of animal milk whichtheingestion of animal milk which

contains microorganisms derived from: contains microorganisms derived from:

a.a. Animal Animal e.g.e.g. by contamination with its by contamination with its feces feces

b.b. The environment The environment

c.c. Milk handlers such as dairy workers Milk handlers such as dairy workers

Introduction:

Importance of milk examination for Importance of milk examination for pathogenspathogens

It is important to examine It is important to examine milk for pathogens to ensure milk for pathogens to ensure that it is safe to be that it is safe to be consumed by man.consumed by man.

Milk is further used for Milk is further used for obtaining many milk obtaining many milk products like products like cheese ,cream , butter and cheese ,cream , butter and ice cream ice cream

E.coli Streptoccus pyogenesMycobacterium bovisBacillus anthracis Salmonella sp. Brucella sp.

Pathogenic bacteria present in Pathogenic bacteria present in milkmilk

Determination of viable Determination of viable bacterial count:bacterial count:

Using the pour plate method after preparation of 10Using the pour plate method after preparation of 10fold serial dilution from the milk sample with ringerfold serial dilution from the milk sample with ringersolution. solution.

Permissible number of bacterial flora in pasteurized milk is 5 x 104 cfu/ml

Permissible number of bacterial flora in long life milk is 10 cfu/ml

Methylene Blue Reduction Test

To determine quality of the milk Increasing the number of bacterial flora will

reducethe color of methylene blue more rapidly due

toincreasing consumption of oxygen.i.e.: The speed of reduction of methylene blue

color is directly proportional to the number of bacteria present

in milk sample.

Methylene Blue Reduction Test

Results:

The shorter the decolorization time, the The shorter the decolorization time, the higherhigher

the number of bacterial flora present in the number of bacterial flora present in milk,milk,

and and the poor quality of milkDecolorization time Result

30 min – 2 hrs Poor quality 2 – 6 hrs fair quality 6 – 8 hrs good quality

Over 8 hrs excellent quality

Test for coliforms

Done by inoculation of MacConkey’s broth with 0.1 ml of milk sample.

Examine for the production of acid detected by changing the color of the medium from purple to yellow.

+ve result with gas production

-ve result

Water

Milk

Air

Examination of water, milk Examination of water, milk and airand air

Importance of keeping the micro- Importance of keeping the micro- organisms count low in airorganisms count low in air

Surgical theatersSurgical theaters

Food preparationsFood preparations

Drug materials Drug materials

Cross infection and out Cross infection and out breaks in hospitalsbreaks in hospitals

Number on bacteria in air depends Number on bacteria in air depends on on

Number of personsNumber of persons

Body movementBody movement

Disturbance of clothingDisturbance of clothing

Methods of examination of airMethods of examination of air

a. Settle platea. Settle plate: : Petri dishes containing an agar medium are left open for a Petri dishes containing an agar medium are left open for a

measured period of time. measured period of time. Large bacteria-carrying dust particles settle on the medium. Large bacteria-carrying dust particles settle on the medium. The plates are incubated and a count of the colonies is The plates are incubated and a count of the colonies is

formedformed

Blood agar is suitable for Blood agar is suitable for over all countover all count

For detection of a For detection of a particular microorganism particular microorganism suitable media is used .suitable media is used .

Disadvantage of this method :Despite its simplicity it measures only therate of deposition of large particles fromthe air

b. Slit samplerb. Slit sampler It draws in air from the environment at a fixed rate and It draws in air from the environment at a fixed rate and

causes the suspended particles to fall on the surface of causes the suspended particles to fall on the surface of the agar plate.the agar plate.

c. Air centrifugec. Air centrifuge

Centrifuging particles from the air on to a Centrifuging particles from the air on to a culture medium. culture medium.

The sampled air passed along a tube lined The sampled air passed along a tube lined with nutrient agar which was rotated on its with nutrient agar which was rotated on its long axis.long axis.

After sampling the strip is removed from After sampling the strip is removed from the instrument and incubated then the instrument and incubated then colonies can be counted.colonies can be counted.

Notice:Notice:

NoNo level of contamination however low level of contamination however low can be regarded as certainly safe.can be regarded as certainly safe.

Infection can be initiated by deposition of Infection can be initiated by deposition of a single infected particle at a favorable a single infected particle at a favorable site.site.

The probability of S. aureus initiated The probability of S. aureus initiated infection is low in comparison with infection is low in comparison with Mycobacterium tuberculosis Mycobacterium tuberculosis

Demonstration:Demonstration:

Air examinationAir examination Settle plate Settle plate Water examinationWater examination Determination of MPN Determination of MPN Milk examinationMilk examination Methylene blue reduction testMethylene blue reduction test