SEMINAR ON MICROBIAL TECHNIQUE

SEMINAR ON MICROBIAL TECHNIQUES USED IN TESTING OF WATER & AIR.:

By, Kasturi panda M . Pharm SEMINAR ON MICROBIAL TECHNIQUES USED IN TESTING OF WATER & AIR.

INTRODUCTION.:

INTRODUCTION. Water is elixir for life. It is an essential part of protoplasm & creates a state for metabolic activities to occur smoothly. No life can exists without water. Major areas (about ¾ ) of earth surface is covered by water , mainly by oceans, & to some extent by lakes , river , streams, ponds etc. water receives micro-organism from air, soil, air, sewage, organic wastes, dead plants & animals etc. Types of water- 1-atmospheric water. 2-surface water. 3-stored water. 4-ground water.

WATER MICRO-ORGANISM:

WATER MICRO-ORGANISM A large no of micro-organism are found in water like fungi, bacteria, protozoa. some of the aquatic fungi in different water bodies are-Rhizophydium sp., Achlya americana., A. debaryana, A. laevis. Some of the water borne pathogenic microbes are- Bacteria- Mycobacterium tuberculosis, Pseudomonas aeroginosa, Salmonella enteriditis. Protozoa- Giardia lamblia, Entamoeba histolytica, Cryptosporium. Microbiological analysis of water purity. The Faecal coliform-these are intestinal bacteria which are present in water due to faecal contamination. Coliform are mambers of family Enterobacteriaceae which includes E. coli, Enterobacter aerogens, Salmonella & Klebsiella pneumoniae. If such organism are found in water it is not suitable for drinking.

Sanitary test for coliform. :

Sanitary test for coliform. The original test for the presence of coliform in water is done by standard multiple tube fermentation techniques. These are 1-The presumptive test. 2-The complete test. Presumptive Test- A series of fermentation tubes each containing lactose broth or lauryl tryptose broth of known conc. are incubated with known amount of water. The tubes are incubated for 24 to 48 hr at 35 c. Here 15 tubes are used,5 tubes with 10 ml, 5 with 1 ml, 5 with 0.1ml. At the end of incubation , the indicates the presence or absence of coliform. Complete test – This test is to ascertain about the presence of coliform in water.Each colonies from positive confirmed test is transferred to lactose fermentation tube and to nutrient agar slants. The tubes are incubated at 35 ˚ c for 48 hr. Production of gas in the fermented tubes shows positive test.

The most probable no of (MPN) of coliform:

The most probable no of (MPN) of coliform MPN is used to calculate the no. of coliform micro-organism in 100ml of water. Techniques used are- 1- Membrane filtration techniques - it is also known as millipore filter tech. A sterile membrane filter (0.45 µm) disk is placed in the sterilized holding apparatus. A volume of water is passed through filter disk. Bacteria present in the water sample are retained in filter disk. The sides of the funnel & membrane are rinsed with sterile water. Thereafter the membrane filter disk is aseptically removed & placed on adsorbent disk saturated with cultural media & contained in petridish. The medium passes through the pores of membrane & nourish the bacteria present in it.After incubation at 35°c for 24hr each bacterium multiplies to form visible colony, which can be easily counted.

MPN of Coliform.:

MPN of Coliform. 2- The defined substrate test- it is used for both coliform & E. coli in a single 100ml water sample. A specialized medium containing O-nitrophenyl-beta-D-galactopyranoside(ONPG) & 4- methylumbelliferyl-beta-D-glucuronide (MUG) is added with water & incubated for 24 hr at 35 °c. If coliform are present the medium will turns yellow within 24 hr due to breakdown of ONGP.When E. coli are present, the MUG is changed to yield a fluorescent product. The fluorescence is observed under long wavelength UV light for the presence of E. coli. 3 -IMViC test –the IMViC represents indole, methyl red, Voges-Proskauer & citrate test. This test is used to distinguish between E. coli & Aerobacter aerogens .

Characteristic IMViC reaction on E. coli & A. aerogens:

Characteristic IMViC reaction on E. coli & A. aerogens Test E. coli A. aerogens Indole Indole is produced fromtryptophan(+) Indole is not produced(-) Methyl red E. Coli produces acid that drops pH below 4.5.hence methylred turned red(+). Methyl red remains yellow due to production of less acid (-). Voges-Proskauer E. Coli does not produce acetyl-methylcarbinol in glucose-peptone medium (-). Acetylmethylcarbinol is produced by this bacterium(+) Citrate Citrate does not support the growth of E. coli.(-) Citrate support the growth of bacterium(+).

Purification of water (water treatment).:

Purification of water (water treatment). There are several methods for purification of water the use of which depends upon amount & quantity of water. There are three chief methods which are used for the purification of water. 1-Sedimantation. 2-Filtration. 3-Disinfection.

Testing compressed airlines for microbial contamination:

Testing compressed airlines for microbial contamination A micro-organism that is capable of forming a viable colony forming unit (CFU) & which exist within the compressed air lines system is called the microbiological particle (MP) as per ISO. The microbial load on a product is referred to as BIOBURDEN. The bioburden came from different sources such as human contact, air conditioning system, the manufacturing process itself, raw materials etc. Routinely compressed air lines should be tested on a routine basis in order to determine the microbiological cleanliness of the air suppply.

Test equipment.:

Test equipment.

Methodology.:

Methodology. The slit sampler (also known as slit-to agar sampler) is a device which utilizes a rotating stage which holds the petri-plate. The air impacts the surface of agar with whatever organism are present, the organism became impinged onto the agar, the plate is incubated, & the organism are allow to grow. Care should be be taken to insure that the equipment used has been properly disinfected in order to minimize the introduction of bacteria not associated with the compressed air supply. A negative control should be used as a qualitative measure, and the end point to be tested should be purged & aseptically cleaned to minimize false result.

Slide 12:

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