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Government College University Faisalabad
COLLEGE OF PHARMACY
Pharm. D 4th semester (evening)SESSION 2007-2012
MICROBIOLOGY II
TO:Assistant Professor HADAYAT RASOOL
CONTENTS
ANTIBIOTICS---introduction
CEPHALOSPORIN Introduction Fermentation
EXTRACTION
LYOPHILIZATION
PACKAGING AND LABELLING
ANTIBIOTICS
INTRODUCTION
ANTIBIOTICSDEFINITION:
Antibiotics are substances that kill or inhibit growth of
microorganisms but causes little or no damage to the host.
ORAntibiotics are antimicrobial agents produced naturally by
other microbes (usually fungi or bacteria).
HOW ANTIBIOTICS COME?
Bacteria cause infections in1. Humans2. Other higher organisms
Point to be noted
How other plants, animals and fungi protect themselves?
HISTORY
The first antibiotic was discovered in 1896 by Ernest Duchesne
and "rediscovered" by
Alexander Flemming in 1928 from the filamentous fungus
Penicilium notatum.
HISTORY
Ernest Duchesne
A French physician
Discovery: thirty-two years before
Alexander Fleming discovery
Entered “The Military Health Service School of Lyon” in 1894
HISTORY
Ernest Duchesne Thesis:
Contribution to the study of vital competition in micro-organisms
▪ antagonism between molds and microbes Submitted in:
In 1897 get his doctorate degree
Important:First study of therapeutic capabilities of molds due to their anti-microbial activity.
HISTORY
Ernest DuchesneObserve:
Arab stable boys
at the army hospital
kept their saddles in a dark and damp room
to encourage mold to grow on them
HISTORY
Ernest Duchesne
Asked , why ?
To heal the saddle sores on
the horses
HISTORY
Ernest DuchesneAchievement no. 1:
Prepared a solution of the mold
Injected it into a series of diseased guinea pigs.
All recovered
HISTORY
Ernest DuchesneAchievement no. 2:
Made interaction between
bacteria (Escherichia coli) and fungus (Penicillium glaucum)
Fungus eliminated bacteria from medium
HISTORY
Ernest DuchesneBad Luck:
1. He was 23 and unknown
2. His army service after getting his degree prevented him from doing any further work
3. Failed to report a connection between the fungus and a substance that had antibacterial properties
4. died at the age of 37
HISTORY
Alexander Flemming
in 1928 Accidentally Discovered penicillin
from the filamentous fungus
Penicilium notatum.
HISTORY
Penicillin was not purified until the 1940s by
Florey and Chain
just in time to be used at the end of the second world war.
HISTORY
Alexander Flemming and Florey and Chain
Share Nobel prize
In 1945
CEPHALOSPORIN
INTRODUCTION, CLASSIFICATION, PROPERTIES AND PRODUCTION
INTRODUCTION
INTRODUCTION
DEFINITION:
Any of various broad-spectrum B-Lactam antibiotics, closely
related to the penicillins, that were originally derived from the
fungus Cephalosporium acremonium.
INTRODUCTION
HISTORYCephalosporin compounds were
first isolated from cultures of
Cephalosporium acremonium
in 1948 by Italian scientist Giuseppe Brotzu
INTRODUCTION
HISTORY
He noticed that these cultures produced substances that were effective against
Salmonella typhi, the cause of typhoid fever,
which had beta-lactamase.
INTRODUCTION
ACTION:
Inhibition of cell wall synthesis
INTRODUCTION
Inactive Against:
1.Enterococci spp. Intrinsic factor
2.MRSA additional substances
3.Legionella spp. Intrinsic factor
4.Mycoplasma spp. No cell wall
5.Chlamydia spp. Intrinsic factor
MRSA= Methicillin-resistant Staphylococcus
aureus
INTRODUCTION
Common Use:
In surgical procedures
to reduce the risk of post operative infections
CLASSIFICATION4 GENERATIONS
CLASSIFICATION
Based Upon:
The spectrum of antimicrobial activity
Grouped w.r.t increased gram –ive and decreased gram +ive
activity,as
CLASSIFICATION
First Generation Examples:
Cefazolin Cephalexin
Spectrum: Most gram positive cocci (Strep, S. aureus) E. coli Proteus Klebsiella.
Use: S. aureus infection, surgical prophylaxis.
CLASSIFICATION
Second Generation Examples:
Cefoxitin Cefuroxime Cefaclor Cefprozil
Spectrum: Mainly effective against G- bacteria Modest activity against G+ bacteria
Use: primarily for Upper Respiratory Tract Infections Lower Respiratory Tract Infections
CLASSIFICATION
Third Generation Examples:
Ceftriaxone Cefotaxime
Spectrum: enhanced G- activity
Use: Meningitis highly resistant and multi drug resistant
strep pneumo along with vancomycin.
CLASSIFICATION
Fourth GenerationExamples:
CefepimeSpectrum:
Active against Strep, Staph aerobic gram negatives
Cephalosporium acremoniumINTRODUCTION
Cephalosporium acremonium
Important To Learn:
IsolationCharacteristics
Colonial characters Microscopic characters
▪ Without staining▪ With staining
ISOLATION
SAMPLECephalosporium acremonium
Most common in 1. Soil2. Plant debris3. Rotting mushrooms
Found in1. Europe2. Asia3. Egypt4. North and Central America
CHARACTERISTICS
Colonial: Colour: pink Moist Gradually become more hyphal
Microscopic: Narrow hyphae Phialides Conidia
▪ Narrow▪ Cylindrical
Arthrospores
PRODUCTIONINDUSTRIAL SCALE
PRODUCTION
STEPS:
1. Fermentation2. Extraction3. Lyophilization4. Packaging and Labelling
SHAKE FLASK CULTURE METHOD
METHOD OF FERMENTATION
FERMENTATION
DEFINITION
Drug producing microbe is grown and cultivated in appropriate culture
vessels or in large fermentors on a suitable medium in which respective
drug is accumulated.
FERMENTATION
STEPS1. Isolation of microbe2. Characterization of microbe3. Purification of culture4. Preparation of inoculum5. Quality control6. Lab scale fermentation7. Quality control8. Seed cultivation9. Quality control
ISOLATION
SAMPLECephalosporium acremonium
Most common in 1. Soil2. Plant debris3. Rotting mushroomsFound in4. Europe5. Asia6. Egypt7. North and Central America
ISOLATION
SABOURAUD'S AGAR1. Glucose-------------
40g
2. Peptone-------------10g
3. Agar-----------------15g
4. H2O (dist)-q.s—1000ml
Filter sterilization
Autoclave
sterilization
ISOLATION
SAMPLE
STERILIZED MEDIUM
CONDITIONS1. TEMP= 25-28oC2. 7 Days3. Humidity
FERMENTATION
STEPS Isolation of microbe Characterization of microbe Purification of culture Preparation of inoculum Quality control Lab scale fermentation Quality control Seed cultivation Quality control
CHARACTERISTICS
Colonial: Colour: pink Moist Gradually become more hyphal
Microscopic: Narrow hyphae Phialides Conidia
▪ Narrow▪ Cylindrical
Arthrospores
FERMENTATION
STEPS Isolation of microbe Characterization of microbe Purification of culture Preparation of inoculum Quality control Lab scale fermentation Quality control Seed cultivation Quality control
PURIFICATION
Malt extract agar (Blakeslee's formula)
1. Malt extract...............20.0 g2. Glucose.....................20.0 g3. Peptone......................1.0 g4. Agar........................20.0 g5. Distilled water..............1.0 L
Autoclave at 121C for 15 minutes Add glucose separately sterilized
MacCorteny bottle
PURIFICATION
Other media used for cephalosporins are1. Potato dextrose agar2. Potato carrot agar
Conditions:1. TEMP= 25-28oC
2. 7 Days3. Humidity
Repeated growth on these media leads to purified culture.
FERMENTATION
STEPS Isolation of microbe Characterization of microbe Purification of culture Preparation of inoculum Quality control Lab scale fermentation Quality control Seed cultivation Quality control
CHARACTERISTICS
Colonial: Colour: pink Moist Gradually become more hyphal
Microscopic: Narrow hyphae Phialides Conidia
▪ Narrow▪ Cylindrical
Arthrospores
PREPARATION OF INOCULUM
5ml water + glass beeds
Pour in
MacCorteny Bottle
ShakePour out
Inoculum
FERMENTATION
STEPS Isolation of microbe Characterization of microbe Purification of culture Preparation of inoculum Quality control Lab scale fermentation Quality control Seed cultivation Quality control
QUALITY CONTROL
Check No. of spores in inoculum
FERMENTATION
STEPS Isolation of microbe Characterization of microbe Purification of culture Preparation of inoculum Quality control Lab scale fermentation Quality control Seed cultivation Quality control
SHAKE FLASK CULTIVATION
"SEED STAGE" MEDIUM(per flask)
1. Corn steep liquor 0.5% nitrogen Lactose ……...……………46 g/l
2. Glucose …………………………………….2 g/l 3. Methionine ……………………………..2.3 g/l 4. Phenyl acetyl ethanbolamine …….1.5 g/l 5. Calcium carbonate ……………………16 g/l 6. Urea ………………………………………0.8 g/l 7. Ammonium sulphate ………………..3.4 g/l 8. Maize oil ……………………………….6 drops 9. Salts solution…………………………… 1 ml/l10. Distilled water ………..q.s…………….1000L
SHAKE FLASK CULTIVATION
Salt solution:1. Fe (NH 4 ) 2 (SO 4 ) 2 .6H 2 O ......15 g/100 ml 2. Mn SO 4 .4H 2 O ........................3 g/100 ml 3. Zn SO 4 .7H 2 O .........................3 g/100 ml 4. Cu SO 4 .5H 2 O ......................0.8 g/100 ml Conditions: 1. 25° C2. 250 rpm3. ph 6.54. 72 hours
FERMENTATION
STEPS Isolation of microbe Characterization of microbe Purification of culture Preparation of inoculum Quality control Lab scale fermentation Quality control Seed cultivation Quality control
Quality control
Sampling After every 8 hours1. Contents examination
Morphological properties of biomass Average no of spores Occurrence of pellet
2. pH 3. Nutrients concentration
Production Analysis
Qualitative Test
Take a strip
Dip in 0.2% bromophenol
2% of sample
dried
Production Analysis
Loopful of beta-lactam resistant strain
Beta-lactamase
Denaturation of lactam ring
Blue to yellow
PRODUCTION ANALYSIS
QUANTITATIVE TESTDisc soaked in cephalosporin sample
and standard solution
Placed on Mueller Hinton Agar
Diameter of inhibition zone of resistant strain
calculation
PRODUCTION ANALYSIS
Mueller Hinton Agar1. 30.0% beef infusion2. 1.75% casein hydrolysate3. 0.15% starch4. 1.7% agar
pH adjusted to neutral at 25 °C.
FERMENTATION
STEPS Isolation of microbe Characterization of microbe Purification of culture Preparation of inoculum Quality control Lab scale fermentation Quality control Seed cultivation Quality control
Seed cultivation
In huge fermentors of capacity 10L to 1000L
FERMENTATION
STEPS Isolation of microbe Characterization of microbe Purification of culture Preparation of inoculum Quality control Lab scale fermentation Quality control Seed cultivation Quality control
Quality control
Sampling After every 8 hours1. Contents examination
Morphological properties of biomass Average no of spores Occurrence of pellet
2. pH 3. Nutrients concentration
Production Analysis
Qualitative Test
Take a strip
Dip in 0.2% bromophenol
2% of sample
dried
Production Analysis
Loopful of beta-lactam resistant strain
Beta-lactamase
Denaturation of lactam ring
Blue to yellow
PRODUCTION ANALYSIS
QUANTITATIVE TESTDisc soaked in cephalosporin sample
and standard solution
Placed on Mueller Hinton Agar
Diameter of inhibition zone of resistant strain
calculation
PRODUCTION ANALYSIS
Mueller Hinton Agar1. 30.0% beef infusion2. 1.75% casein hydrolysate3. 0.15% starch4. 1.7% agar
pH adjusted to neutral at 25 °C.