MICROBIOLOGICAL ASSAYS

PRESENTED BY K.SWETHA

MICROBIOLOGICAL ASSAYS

BIOLOGICAL ASSAY REFERS TO MEASUREMENT OF THE RELATIVE

POTENCY OR ACTIVITY OF
COMPOUNDS BY DETERMINING THE AMOUNT REQUIRED FOR PRODUCING AN ANTICIPATED EFFECT ON A SUITABLE TEST ANIMAL OR ORGAN UNDER STANDARD CONDITIONS.

MICROBIAL ASSAY IS A TYPE OF BIOLOGICAL ASSAY PERFORMED WITH MICROORGANISMS.

E.g:- BACTERIA,YEAST AND MOLDS.

MICROBIOLOGICAL ASSAY OF ANTIBIOTICS

It

is based upon a comparision of the inhibition of growth of bacteria by measured concentration of the antibiotic under test with that produced by a known concentration of a standard preparation of the antibiotic having a known activity.
The

microbiological assays of antibiotics are based on either (a) serial dilution method or (b) diffusion method.

SERIAL DILUTION METHOD

The dilution of the antibiotic under test thatll inhibit growth of a susceptible organism is compared with dilution of a standard preparation that has identical effect. Strength of the unknown can be calculated from the knowledge of the potency of the standard. Dilution in broth is suitable but it is more satisfactory in nutrient agar medium.

DIFFUSION METHOD

Preferred over serial dilution method. Dilutions of the antibiotic under test and standard are made in geometric proportions. Plates are prepared by pouring agar,seeding them with test organism,and allowing to set on a horizontal surface.

Plates are left at room temp. for 2 hrs to allow diffusion of antibiotic solution throughout the medium to get ahead of growth of the organism.
After incubation, inhibition of growth can be observed as a clear zone of inhibition around each container.

STANDARD CURVE
It is plotted between log concentration Of standard against zone of inhibition,and log conc. Of test against zone of inhibition. If two lines are parallel, relative potency of standard and test are represented by horizontal dist between the lines.

OFFICIAL ASSAY METHODS

Two recommended methods for antibiotic assay by pharmacopoeia are : - cylinder plate method - turbidimetric method Following considerations are common to the above methods : - standard preparation and units of activity - test organism and inoculum - apparatus - assay designs

MICROBIOLOGICAL ASSAY OF VITAMIN B12 ( CYANOCOBALAMIN) :

Basic medium quite complex (contains a variety of essential components as a mixture in solution). One set of tubes contains measured amounts of standard cyanocobalamin solution and another set of tubes with graded volumes of test sample. All the tubes are inoculated with a small amount of culture of Lactobacillus leichmannii and then incubated. The extent of growth is determined by measuring light transmittance in a spectrophotometer.

ANTIBIOTICS

ANTIBIOTICS
Antibiotics are medicines that kill bacteria or slow the growth of bacteria. They are used to cure diseases. Antibiotics are natural substances that are released by bacteria and fungi into the their environment. In 1928, Sir Alexander Fleming observed that colonies of the bacterium Staphylococcus aureus could be destroyed by the mold Penicillium notatum and this lead to the discovery of pencillins.

TYPES OF ANTIBIOTICS
Penicillins Cephalosporins Aminoglycosides Macrolides Sulfonamides Fluoroquinolones Tetracyclines Polypeptides

PENICILLINS

Penicillins are n-acyl derivatives of 6-aminopencillanic acid (6APA). Most penicillins are produced by species of Penicillium while others are semi synthetic. It is produced from Penicillium notatum and Penicillium chrysogenum.

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MECHANISM OF ACTION
Penicillins

are active against many gram positive bacteria,Nocardia and Actinomyces, but not against most Gram negative forms except at higher dosage levels.
It interferes with cell wall synthesis of sensitive organisms and is active only against growing cells.

BIOSYNTHESIS OF PENICILLIN IN
PENICILLIUM CHRYSOGENUM

PRODUCTION OF PENICILLIN

The Mould Inoculum Preperation Fermentation Extraction and Purification

a) removal of mycelium b) countercurrent solvent extraction of pencillin c) treatment of crude extract

PRODUCTION OF PENCILLIN.
Mould:

High yielding strains developed from Pencillium chrysogenum ,obtained either by mutation or recombination ,are used.

Inoculum Preparation: Spores from working stock are added to

nutrient medium & incubated for 5-7 days at 24C. The resulting spores are transferred into inoculum tanks, incubated for 1-2 days. The resulting inoculum is used for a production tank ,to produce inoculum for larger scale fermentations.
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FERMENTATION:
DAYS.

PENICILLIN FERMENTATION IS INCUBATED AT 25-26C FOR 3-4

Production medium(Jackson medium):

Corn steep liquor Lactose Glucose CaCO3 KH2PO4 Edible oil Phenyl acetic acid pH after sterilization 3.5% 3.5% 1% 1% 0.4% 0.25% Precursor 5.6----6

Extraction & Purification:

Removal of mycelium: by filtration. Contercurrent solvent extraction of Penicillin. Treatment of crude extract
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THE COURSE OF A TYPICAL PENCILLIN FERMENTATION IS PRESENTED IN THE FOLLOWING FIG:

FLOW

SHEET FOR LARGE SCALE PRODUCTION OF

PENICILLIN-G :

SEMISYNTHETIC PENCILLINS

The semi synthetic penicillins contain 6-aminopenicillanic acid(6APA) nucleus. 6-APA is prepared on large scale by removal of the phenyl acetate side chain from penicillin-G. Enzyme acylases of microbial origin is used for this purpose.

Enzyme penicillin amidases (acylases):

This enzyme catalyses the removal of the side chain from benzyl penicillin. It is a membrane bound enzyme found in E.coli and Alcaligenes. It has high specificity for penicillin G. 6-APA is used for the production of new penicillins eg:Methicillin,naficillin,and isoxazolyl penicillins, the broad spectrum penicillins ,amoxicillin,epicillin,and cyclacillin;antipseudomonal penicillins such as carbenicilin and piperacillin,azocillin and mezlocillin.

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STREPTOMYCIN
Streptomycin is produced by Streptomyces griseus. It is an important chemotherapeutic agent active against gram positive and gram negative bacteria including mycobacterium.

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BIOSYNTHESIS OF STREPTOMYCIN

PRODUCTION OF STREPTOMYCIN:
Inoculum:

High yielding mutated Streptomyces griseus spores are maintained as soil stocks or lyophilised in a carrier such as sterile skim milk.

Fermentation process:
Culture conditions:

Temperature:25-30C(approximately 28C) pH:between7 and8(peak production7.6-8) Fermentation period :5-7 days. Yield in production vessel strongly responds to aeration and agitation condition.

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MEDIUM:
WOODRUFF & MC DANIEL
Soya bean meal----1% Glucose----1% NaCl----0.5%

HOCKENHULL MEDIUM
Glucose----2.5% Extracted soya bean meal---4% Distillers dried solubles0.5%

NaCl--------0.25%
pH (before sterilization)---7.3 to 7.5

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FERMENTATION PROCESS:

1st PHASE: It lasts approx. 24hrs with rapid growth during this period. The strong proteolytic activity of Streptomyces griseus releases ammonia to the medium from the soyabean meal & the carbon nutrients of the soya bean meal are utilized for growth. During this period the PH of the medium rises from approx. 6.7 or 6.8 to 7.5 or slightly higher. 2nd PHASE: During this phase streptomycin is produced at rapid rate, lasts from approx. 24hrs to 7 days of incubation. No mycelial growth occurs and thus the weight of mycelium remains fairly constant. Ammonia is utilized and the glucose is rapidly withdrawn from the medium so that little is left by the end of this phase. The ph remains in a range of 7.6-8. IN the last phase,PHASE 3, the sugar has been depleted from the medium and streptomycin production ceases The cells lyse, releasing NH3 and the PH rises.

ERYTHROMYCIN

For industrial production of Erythromycin , the producing genera belongs to Streptomyces(S.erythraeus,S.griseoplanus),and Micromonospora. In the erythromycin fermentation, erythromycin A is usually produced as the main product, with erythromycin B,C,and D as minor components.

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PRODUCTION OF ERYTHROMYCIN
Inoculum preparation:

To obtain stable inoculum, atleast 10*8 viable cells/ml of suspension are necessary. Sporulation is carried out on tryptone agar slant. After harvesting ,the cells are preserved at 4 degrees C. Medium: Culture conditions: Fermentation tempLength -3 to 7 days 33 C

Sucrose------- 5% CSL-----------0.5% Soya bean oil meal------1.5% Yeast---------------1% NaCl----------------0.5% CaCo ppt---------0.3% pH-------------------7-7.2

FERMENTATION:

Fermentation is carried out in a stirred tank fermenter. Production of erythromycin occurs when growth reaches the stationary phase.

Isolation and Extraction:

Isolation: Mycelium is separated from the broth by filter press,centrifuge or drum filter with a filter aid. Extraction: using a water immiscible organic solvent(methyl isobutyl ketone or ethyl acetate) It is then transferred to acidic water and the pH is adjusted with HCl,H3PO4,acetic acid or citric acid.

Purification and concentration : Is carried out

with ion exchange resin i.e.; amberlite-50. The antibiotic absorbed on resin is eluted by a mixture of organic solvent and water at 3---8 pH.

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ENZYMES
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AMYLASES

Amylases are enzymes which hydrolyse starch. The hydrolysis of starch with amylase results first in the production of short chain polymers called dextrins,then the disaccharide maltose and finally glucose.

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USES:
Production of sweetners in food industry. Production of bread. Brewing industry. Dry-cleaning industry. Liquefaction of heavy starch pastes formed during heating steps in manufacture of corn & choc syrup.

-AMYLASES

-amylases (1,4--glucan-glucanohydrolases) are extracellular enzymes which hydrolyse 1,4-glycosidic bonds. These are endoenzymes. -Amylases are formed by many bacteria(B.subtilis,B.polymyxa, Micrococcus) and fungi(Aspergillus,Pencillium,Cephalosporium) The molecular weights of various -amylases do not differ considerably. They all contain a large proportion of tyrosine and tryptophan in the enzyme protein & most require Calcium as stabilizer

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PRODUCTION OF A-AMYLASES
BACTERIAL -AMYLASES Regulated by several genes, which have been only partially characterised. Starch: NH4NO3: Na.citrate: K2HP04: MgSO4.7H2O: CaCl2.2H2O: peptone: yeast extract pH 0.5% 0.56% 0.28% 0.13% 0.05% 0.01% 0.5% 0.2% 6.8. FUNGI -AMYLASES

o o o o o o o o o o

o o o o o o o o o

Production is Constitutive. starch: 8% K2HPO4: 0.1% NaNO3: 1.2% MgSO4: 0.1% KCL: 0.05% FeSo4: 0.03% Mg(NO3)2: 0.08% Mg(H2PO4)2: 0.05% Malt Extract: 2% The optimal temperature: 28 to 30 degrees C Strain used:Aspergillus oryzae
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Optimal temperature:27 to 30C. Strain used: Bacillus subtilis

-AMYLASES

-Amylases(-1,4-glucan-maltohydrolases) are usually of plant origin, but some microbial producers are also known: Bacillus polymyxa,B.cereus,Streptomyces sps,Pseudomonas sp,and Rhizopus japanicus. Bacterial -amylases have greater heat resistance(>70C) than plant -amylases and the pH optimum is also higher (about pH 7). In contrast to -amylase ,calcium is not necessary for stabilisation and activation of -amylase.
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GLUCOAMYLASES

Glucoamylases(-1,4-glucan-glucohydrolases) act on starch by splitting glucose units from the nonreducing end. Glucose ,maltose and limit dextrins are the end products of glucoamylase action.
Microorganisms used to produce glucoamylases are Aspergillus niger,A.oryzae,Rhizopus niveus,R.javanicus.
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REFERENCES:
Pharmaceutical Biotechnology:
Fundamentals and Applications (S.S Kori;M.A.Halkani) A Textbook of Industrial Microbiology (Wulf Crueger and Anneliese Crueger) Industrial Microbiology (L.E.Casida,JR) Industrial Microbiology (A.H Patel)