313 PHT Lab. No. 8

Aerobic, non-fermentative, motile, oxidase-positive gram- negative bacilli. Aerobic, non-fermentative, motile, oxidase-positive gram- negative bacilli. Most Important Species Most Important SpeciesP.aeruginosa  opportunistic pathogen  causes UTI, wound infections and otitis media Pseudomonas

Microscopical examination: (morphology) A) Gram’s Stain: Gram –ve Non-sporeforming bacilli, having single arrangement.

B) Examination of Motility: Using the “Hanging Drop technique” Pseudomonas is highly motile by means of polar flagella. Pseudomonas is highly motile by means of polar flagella.

Cultural characteristic: It grows on simple media. It usually produces exopigments. 1) Growth on nutrient agar: Its growth on nutrient agar showing greenish discolouration due to exopigment production.

 Cetrimide agar is a highly selective medium for pseudomonas species due to presences of cetrimide which inhibits the growth of other bacteria.  It contains also MgCl 2 & K 2 So 4 to facilitate production of the charactaristic green pigment of pseudomonas. 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 enteric gram –ve bacteria (bile salt inhibit the growth of non enteric bacteria).  Test sugar: lactose.  pH indicator: neutral red ( yellow in alkaline, pink in acidic pH). 3) Growth on MacConkey’s agar: Principle:

Biochemical reaction: 1)Oxidase test. 2) Nitrate 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 test Pseudomonas No colour -ve test Enterobacteriaceae 1)Oxidase test:

2) Nitrate Test: Principle: Nitrate Nitrate reductase nitrite α -naphthyl amine (nit. A) Sulphanilic acid (nit. B) Red diazonium saltEnterobacteriaceae Further reduction Nirtogen (N 2 ) 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 35 o C for 24 hrs

Results: Red colour after addition of nit.A & nit.B Reduction of Nitrate to nitriteEnterobacteriaceae Red colour after addition of zinc dust -ve reduction No red colour after addition of zinc dust Further reduction to Nitrogen Pseudomonas

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

Positive Test: O - /F - O + /F + O + /F - O - /F + Results: FermentativeEnterobacteriaceae Oxidative Pseudomonas Non Saccharolytic

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

Results: 1. No Fermentation: Butt: alkaline (red)Slant: alkaline (red)

Results: Butt: Slant: H 2 S : acidic (yellow) -ve acidic (yellow) alkaline (red) -ve acidic (yellow) alkaline (red) +ve alkaline (red) -ve

Pseudomonas aeruginosa Pseudomonas aeruginosa I'm also very resistant to most antibiotics, so it's very hard to get rid me.

Bacteriological Examination of water, milk and air

Water  Milk  Air Examination of water, milk and air

Importance of water examination for pathogens Water intended for human consumption should not contain any pathogenic organisms. Water intended for human consumption should not contain any pathogenic organisms. Water is used for many applications either at home for cooking,washing or drinking or in industries such as food and pharmaceuticals. Water is used for many applications either at home for cooking,washing or drinking or in industries such as food and pharmaceuticals.

It is also important for hospitals for example haemodialysis unit It is also important for hospitals for example haemodialysis unit Testing of water samples are done regularly to make sure of its safety Testing of water samples are done regularly to make sure of its safety

Supplies of drinking water contaminated with sewage may cause diseases such as: typhoid fever and cholera. Supplies of drinking water contaminated with sewage may cause diseases such as: typhoid fever and cholera. All sources of water should be tested regularly. All sources of water should be tested regularly. Microorganisms which indicate the fecal pollution in water are usually common intestinal commensal bacteria. Microorganisms which indicate the fecal pollution in water are usually common intestinal commensal bacteria.

Most important indicators of fecal pollution of water  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 they Coliforms are members of the enterobacteriaceae family and they 1.grow in the presence of bile salts. 2.produce acid and gas from fermentation of lactose at 37°C. It is the commonly-used bacterial indicator of sanitary quality of food and water. It is the commonly-used bacterial indicator of sanitary quality of food and water.

 Enterococcus faecalis: less numerous than E.coli in human feces, but more resistant to chlorination. less numerous than E.coli in human feces, but more resistant to chlorination.  Clostridium perfringens: Less numerous in human feces Less numerous in human feces Its spores can survive in the environment Its spores can survive in the environment Resist treatment processes than most of the indicators. Resist treatment processes than 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:  Glucose azide broth.  Clostridium perfringes:  Differential reinforced clostridial medium.

 Membrane Filtration Method  Determination of Most Probable Number (MPN) by dilution method  Pour 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 ml DSMB 5 x 10 ml DSMB 5 x 5 ml SSMB 50 ml water sample 10 ml water sample 1 ml water sample

Results:  Positive tubes: showing production of acid or gas.  Acid production: change color of tube from purple to yellow  Gas production: detected in the Durham’s tube.

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

13 2 MPN = 14 i.e: No. of coliform bacilli per 100 ml water sample is 14 cells.

Most probable number of coliforms by McCrady’s table

Using 10 fold serial dilution method Viable Bacterial Count 9 ml Saline 1 23 Water sample 1 ml water1 ml 1/10 1/10 x 1/10 1/100 1/100 x 1/10 1/ ml Melted NA 1 23

Results: Dilution factor 123X X. y 10 x1x1x1x1 X 1.y x2x2x2x2 X 2.y x3x3x3x3 X 3.y 3 No. of colonies per plate Y No. of cells per 1 ml = X 1.y 1 + X 2.y 2 + X 3.y 3 3

 Water Milk  Air Examination of water, milk and air

Human infections may be caused by theingestion of animal milk which contains microorganisms derived from: a. Animal e.g. by contamination with its feces b. The environment c. Milk handlers such as dairy workers Introduction:

Importance of milk examination for pathogens It is important to examine milk for pathogens to ensure that it is safe to be consumed by man. It is important to examine milk for pathogens to ensure that it is safe to be consumed by man. Milk is further used for obtaining many milk products like cheese,cream, butter and ice cream Milk is further used for obtaining many milk products like cheese,cream, butter and ice cream

 E.coli  Streptoccus pyogenes  Mycobacterium bovis  Bacillus anthracis  Salmonella sp.  Brucella sp. Pathogenic bacteria present in milk

Determination of viable bacterial count: Using the pour plate method after preparation of 10 fold serial dilution from the milk sample with ringer solution.  Permissible number of bacterial flora in pasteurized milk is 5 x 10 4 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 reduce the color of methylene blue more rapidly due to increasing 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 higher the number of bacterial flora present in milk, and and the poor quality of milk Decolorization timeResult 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 and air

Importance of keeping the micro- organisms count low in air Surgical theaters Surgical theaters Food preparations Food preparations Drug materials Drug materials Cross infection and out breaks in hospitals Cross infection and out breaks in hospitals

Number on bacteria in air depends on Number of persons Number of persons Body movement Body movement Disturbance of clothing Disturbance of clothing

Methods of examination of air a. Settle plate : Petri dishes containing an agar medium are left open for a measured period of time. Petri dishes containing an agar medium are left open for a 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 formed The plates are incubated and a count of the colonies is formed

Blood agar is suitable for over all count Blood agar is suitable for over all count For detection of a particular microorganism suitable media is used. For detection of a particular microorganism suitable media is used. Disadvantage of this method : Despite its simplicity it measures only the rate of deposition of large particles from the air

b. Slit sampler It draws in air from the environment at a fixed rate and causes the suspended particles to fall on the surface of the agar plate. It draws in air from the environment at a fixed rate and causes the suspended particles to fall on the surface of the agar plate.

c. Air centrifuge Centrifuging particles from the air on to a culture medium. Centrifuging particles from the air on to a culture medium. The sampled air passed along a tube lined with nutrient agar which was rotated on its long axis. The sampled air passed along a tube lined with nutrient agar which was rotated on its long axis. After sampling the strip is removed from the instrument and incubated then colonies can be counted. After sampling the strip is removed from the instrument and incubated then colonies can be counted.

Notice: No level of contamination however low can be regarded as certainly safe. No level of contamination however low can be regarded as certainly safe. Infection can be initiated by deposition of a single infected particle at a favorable site. Infection can be initiated by deposition of a single infected particle at a favorable site. The probability of S. aureus initiated infection is low in comparison with Mycobacterium tuberculosis The probability of S. aureus initiated infection is low in comparison with Mycobacterium tuberculosis

Demonstration:  Air examination Settle plate Settle plate  Water examination Determination of MPN Determination of MPN  Milk examination Methylene blue reduction test Methylene blue reduction test