LABORATORYTECHNIQUES IN .#
R PTIWARI G S HOONDAL RTEWARI
LABORATORYTECHNIQUES IN
MICROBIOLOGY &
BIOTECHNOLOGY • R. P. TIWARI
• G. S. HOONDAL
• R. TEWARI
Departments of Microbiology and Biotechnology, Pan jab University, Chandigarh
I
~ ABHISHEK PUBLICATIONS CHANDIGARH (INDIA)
All nghts reserved No part of this book may be reproduce or transmitted in any form or by any means, electronic or mechanical, including photocoPYing, recording or by any information storage and retrieval system, without permission In wntlng from the publisher. Cover Photographs: Sh. M L Sharma In-charge (Electron Microscope) PU. Chandlgarh- 160014
ISBN :978-81-8247-077-4 ISBN: 81-8247-077-3 Authors First Edition: 2004 Published by : Bharat Shushan Abhlshek Publications 57-59, Sector 17-C, Chandlgarh-17 Ph. : 2707562, Fax: 2704668, Emall:
[email protected] Laser Type by : Outline GraphiCS, Chandigarh Cover Design. R P Verma Printed at . Mehra Offset Press, DaryaganJ, New Delhi
·t~
i
CONTENTS Ex. No
Title
Page
Preface Introduction Bacteriological laboratory safety rules
Unit I: Observation and study of structure of bacteria
2 3
4
5 6
7 8 9 10 II 12 13 14 15 16 17 18 19 20 21 22 23
24 25
26 27
Demonstration of omnipresence of microbes Microscopy: Study, use and care of microscope Examination of microorganisms in live preparations i. Hay infusion examination ii. Examination of protozoa iii. Hanging drop technique iv. Motility in semi ~olid agar Examination of blue green algae (BGA) Preparation of bacterial smear and simple staining Study of morphology of bacteria Observation of capsule and bacteria using negative staining Differential staining of bacteria (Gram's staining) Demonstration of pleomorphism in microorganisms Exam ination of spirochetes Slide culture technique for fungi (microscopic examination) Lacto phenol cotton blue staining for fungi Acid fast staining Staining ofbactcrial spores Capsule staining Demonstration of bacterial cell \vall Demonstration of tlagella in bacteria Demonstration of metachromatic granules in bacteria Demonstration of fat storage globules in bacteria Nucleic acid staining in bacteria Detcrm ination of size of bacteria Differentiation between live and dead microorganisms
Unit II: Microbial physiology: growth and metabolism Preparation of nutrient media Aseptic transfer of culture Isolation of pure culture of bacteria I solation of bactcriophages from sewage Determ ination of viable counts of bacteria Pour plating technique Spread plate method (surface viable counts) Drop method (Milcs and Misra method) MPN method Roll tube technique
9 10
13
16 17 20 22
24 26 27
29 30 32 34 36 38 40
42 44 45 46 48
51 56 59
62 63
28 29 30 31 32 33 34
Study of bacterial growth cycle by determining viable counts Study of bacterial growth cycle by measuring turbidity and biomass determ ination Demonstration of catabolic repression in bacterial culture Effect of oxygen on the growth of bacteria Effect of physical factors (pH and temperature) on growth Study of biochemical characteristics of bacteria Identification of unknown bacteria.
35 36 37 38 39 40 41 42 43 44
Unit III: Bacterial genetics and molecular biology Iso Iation of bacterial mutants Study of mutagens by Ames test Genomic DNA extraction from bacteria Agarose gel electrophoresis for DNA Plasm ids DNA isolation Bacterial transformation Bacterial transduction Bacterial conjugation Estimation of protein by biuret method Estimation of protein by Lowry's method