Preservation of microbes in industry

1. Reaccredited with A+ grade with a CGPA of 3.39 in the III cycle of NAAC
affiliated to manomanium sundaranar university, tirunelveli.
Post graduate & Research Centre – Department of
Microbiology
(government aided)
ACADEMIC YEAR 2021-2023
4TH SEM CORE: FERMENTATION AND INDUSTRIAL MICROBIOLOGY
UNIT-1 PRESERVATION TECHNIQUES
SUBMITTED TO:
DR. S. VISHWANATHAN,
HEAD OF MICROBIOLOGY
DEPARTMENT.
SUBMITTED BY:
A. VASIMA,
REG. NO: 20211232516125
2ND M.Sc. MICROBIOLOGY.

2. Introduction
Objective of
preservation
Types of
preservation
Reference
SYNOPSIS

3.  Isolation, preservation and detection of industrially useful
microorganisms is a time consuming and very expensive process.
Therefore, it is essential to keep the isolated organisms in a viable
condition so that it retains the desirable characters and it can be
used whenever required for industrial production.
 This is done by storing it by creating certain special environmental
conditions by which it remains in a viable condition but in an
inactive state.
 This phenomenon is called as preservation of culture. The
preservation of culture should be done in such a way that it
eliminates the genetic changes, prevents contamination and retains
the viability.
INTRODUCTION

4. OBJECTIVE OF PRESERVATION
To maintain isolated pure cultures for
extended periods in a viable conditions.
To avoid the contamination.
To restrict genetic change[Mutation]

5. WAYS TO PRESERVE INDUSTRIAL CULTURES
Serial
Subculture.
Preservation
under oil
Freeze
drying
Preservation
in distilled
water
Preservation
on soil
Preservation
by liquid
nitrogen

6.  This is the most common, simplest and routine method of
preservation of microorganisms. Selected microorganisms are
initially grown on agar slants. After sufficient growth has taken
place, they are transferred to fresh medium before they lose
their viability.
 There are several factors to be borne in mind while choosing a
suitable medium.
 Solid media should be chosen in preference to liquid media, as
growth of a contaminant can be more readily observed.
SERIAL SUBCULTURE

7.  The appropriate time period for such transfer ranges from a
week to few months (generally four to eight months).
 Preservation of some bacteria by serial subculture,
BACTERIA MEDIUM TRANSFER
TIME
INCUBATION
TEMPERATUE
STORAGE
TEMPERAT
URE
Bacillus spp. Nutrient agar 12 months or
longer
28 10
Pseudomonas
spp.
Nutrient agar 3 months 28 10
Clostridium
spp.
Robertson’s
cooked meat
medium
6 months or
longer
28 Room
temperature.

9. Advantages:
This method is cheap,
Needs no special equipment
Recommended for small collection centers, and
Retrieval easy
Disadvantages:
Change in physiological and genetical characters, and
Time consuming.

10.  This method of preservation is a modification of serial subculture
technique. It was first extensively used by Buell and Weston in 1947.
 This method is very cheap and easy, since it does not require special
apparatus such as a centrifuge, desiccator, or vacuum pump.
 In this method tubes with sterile agar slants are inoculated with a
given culture. The tubes are incubated till sufficient growth of the
given microbe takes place.
 By using sterile technique, a healthy agar slant culture is covered
with a suitable mineral oil to a depth of about 1 cm above the top
of the slanted surface. Thus, over laid cultures can be stored at
room temperature. But better viability is obtained when stored at
low temperature by about 15°C.
PRESERVATION UNDER OIL

11.  The oil used should be of good quality.
 Paraffin oil [of specific gravity 0.865 to 0.890] is generally used
in this method. The oil is sterilized either in McCartney bottles
for 15 minutes at 15lbs pressure or in an autoclave at 15lbs
pressure for 2 hours and then dried in an oven at 170°C for 1-
2 hours, before it is used.
 The covering of the culture with oil prevents drying out.
 The oil allows slow diffusion of gases so growth continues at a
reduced rate.
 Some fungus appear stable and survival of over 20 years by
this method.

13.  It is one of the best methods for long-term preservation of
microorganisms. It is generally used for the preservation of
fungi, viruses, bacteria, enzymes, toxins, sera and other
microorganisms. It is a convenient method for the preservation
of large number of cultures.
 Freeze drying, which is also called as lyophilization involves
freezing of a culture followed by its drying under vacuum
which results in the temporary inhibition of metabolic
activities of microorganisms.
FREEZE DRYING

14. The technique consists of the following stages:
 The organism is allowed to grow to the maximum stationary phase on
a suitable sterilized medium.
 The cells are suspended in a protective medium like milk, serum or
sodium glutamate.
 A few drops of suspension are transferred to a glass ampoule.
 The ampoules are then frozen by immersing into a freezing mixture
of dry ice and alcohol at 78°C and are subjected to high vacuum until
evaporation takes place completely.
 The ampoules are then sealed and stored in a refrigerator.
 The method of revival vary from laboratory to laboratory. Generally,
during revival process the ampoules are decaped under sterile
conditions and the dried pellets consisting of cells of the culture are
transferred to a suitable liquid medium and are allowed to dissolve in
order to make a suspension of cells.
 Then the cells are streaked on to agar plates. Sometimes there may be a
need to undertake repeated sub-culturing for getting a culture
exhibiting all characters. Cells of a lyophilized culture may remain
viable for 10 years or more.

16. Advantages:
 Culture once dried needs no further attention,
 It needs very cheap storage equipment like refrigerator, and
 It is easy to transport freeze-dried ampoules to far off places
in large numbers in relatively small boxes.
Disadvantages:
 This is expensive and needs expertise.

17.  Over 50 years ago, Castellani introduced the concept that fungi could be
preserved in distilled water. This preservation method was extensively
tested on 594 fungal strains, with 62% of the strains growing and
maintaining their original morphology.
 In another study 76% of yeasts, filamentous fungi, and actinomycetes
survived storage in distilled water for 10 years.
 From the margin of well-grown fungal cultures, plugs are cut with sterile
polypropylene transfer tubes and transferred to cryovials containing
approximately 2ml of sterile distilled water.
 Alternatively, Wheaten 4ml glass vials with rubber lined caps may be used.
The vial are stored at room temperature.
PRESERVATION IN DISTILLED
WATER

18.  For many soil born species, survival in soil is necessary.
 Its mainly applied for sporulating microbes
 Survival for 20 years in soil has been reported for Pythium,
Fusarium, and Verticillium spp.
 Fungal cultures are grown on agar medium. A spore
suspension is prepared and pipetted into tubes of dry, sterile
soil[1ml suspension per 5gram of soil] and allowed to dry at
25C. The tubes are stored at room temperature.
PRESERVATION ON SOIL

19. Advantages:
1. Cheap and convenient,
2. Free from mites infestation, and
3. Though some variations may occur but the cultures are stable
and survive upto 10 years.

20.  This method is also called as cryogenic storage method, because a
cryoprotective agent in the form of 10% glycerol is used. Industrially
useful microorganisms are stored under very low temperature ranging from
-150°C – 196°C. In this method ranging, low temperatures are created by
employing liquid nitrogen. Metabolic activities of microorganisms are
reduced considerably at this low temperature. This method is generally
employed for the preservation of fungi, bacteriophages, viruses, algae,
yeasts, animal and plant cells, and tissue cultures.
 This technique involves growing the desired microorganism in sufficient
quantity either in the form of cells or spores or fragments of fungal
mycelium.
PRESERVATION BY LIQUID
NITROGEN

21.  The grown up culture is suspended in 10% glycerol. The
suspension is then introduced in to small ampoules at the rate
of approximately 0.5 ml each. The ampoules are usually made
up of borosilicate glass.
 The ampoules containing culture suspension are frozen and
sealed hermitically. Freezing is done either by directly dipping
the ampoules into the liquid medium or hanging the ampoules
initially over the column of liquid nitrogen for some time and
finally dipping into the liquid.
 The frozen ampoules are then dipped one above the other on
small aluminum containers at the rate of six ampoules per can.
The cans are then packed in aluminum boxes, 20 per each box.
The perforations allow free flow of liquid nitrogen.

23. Advantages:
1. Viable cultures may be preserved for many years by this
method, especially those cultures which do not withstand
preservation by freeze drying.
2. Though the equipment is costly, the process is economical.

24. Books ;
 Industrial microbiology, A. H. Patel,
 Industrial microbiology – Culture preservation and inoculum development,
R. L. Manoghan, M.M. Gagliardi, and S. L. Streicher.
 https://www.biotechnologynotes.com/microorganisms/how-to-preserve-
industrially-useful-microorganisms-top-7-methods-and-techniques/13701
REFERENCE

27. THANKS TO
SRI PARAMAKALYANI COLLEGE