maintenance and transport of cultures maintenance of cultures agar slant culture paraffin method storage in saline suspension preservation in sterile soil preservation by drying in vaccum cryopreservation lyophilization transport media

1. MAINTENACE AND
TRANSPORT OF
CULTURES
Mary Theresa
MSc. Microbiology

2. WHY MAINTENANCE OF CULTURES
NEEDED?
 To isolate bacteria in pure cultures.
 To demonstrate their properties.
 To obtain sufficient growth for the preparation of antigens.
 To determine sensitivity to antibiotics.
 To estimate viable counts.
 Maintain stock cultures.

3. MAINTENANCE OF CULTURES

4. Agar Slant Culture
 Agar slants or stabs- prepared after
autoclaving the medium.
 placing them on slightly inclined positions-
preparing slants- allow to soldify.
 Organism taken from master culture.
 Inoculated with individual culture and
incubated for 24 hours.
 Stored at refrigerator(5°C) or freezer (-20°C).
 Cultures are periodically sub- cultured at an
interval of one to six months.
 Time interval varies with the organisms and
the condition of growth.

5. Paraffin Method/ Preservation by Overlaying
Cultures with Mineral Oil
 Simple, most economical method.
 Agar slants are inoculated &
incubated.
 Then, covered with sterile mineral oil
to a depth of 1cm above the tip of
slant surface.
 Transfers are made by removing a
loop full of growth- touching the tip to
the glass surface to drain off excess
oil- inoculating a fresh medium-
preserving the initial stock culture.
 Functions- providing an aerobic
condition, prevents the dehydration of
the medium and decreases the
metabolic rate of the organisms.

6. Storage in Saline Suspension
 Bacteria- salts for various
metabolic activities.
 But at high concentration, salts act
as bacteriostatic (temporary growth
inhibitor).
 Bacterial culture is preserved in
1% salt concentration in screw-
caped tubes to prevent
evaporation.
 The tubes are stored in room
temperatures.
 Whenever needed the transfer is
made on agar slant.

7. Preservation in Sterile Soil
used in preservation of cultures that form spores.
Prepared by mixing sand and soil- adding small amount of calcium
carbonate- mixture is transferred to screw capped tubes or tubes
plugged with cotton.
Tubes are then autoclaved- thick suspension of spores is added.
Moisture is removed by quickly placing the cap and maintaining
reduced pressure over a drying agent.

8. Preservation by drying in Vacuum
 The cultures are dried over
CaCl2 in a vacuum, then stored
in a refrigerator.
 At such conditions, the
organisms survive for longer
period than the air dried
cultures.

9. Cryopreservation
 Freezing in liquid nitrogen at -
196°C or in the gas phase above
the liquid nitrogen at -150°C.
 In the presence of stabilizing
agents such as glycerol or
dimethyl sulfoxide (DMSO)-
prevent cell damage and promote
cell survival.
 Successful with many species that
cannot preserved by lyophilisation.
 Most species can be remain viable
under these conditions for 10 to 30
years without undergoing change
in their characteristics.
 Method is expensive.

10. Lyophilisation or Freeze- drying
 Process where water and other
solvents are removed from a
frozen product via sublimation.
 The cells in the gas ampoules are
added to the protective agent such
as bovine serum and rapidly
frozen at low temperatures- dried
in high vaccum- ampoules sealed-
stored at low temperatures.
 Most frequently used technique by
culture collection centers.
 Many of the bacterial culture is
preserved by this method have
remained viable and unchanged
their characteristics for more than
30 years.

11. Advantages of Lyophilisation
 Only minimal storage space is required.
 Small vials can be sent conveniently to other microbiological
laboratories when packaged in a special sealed containers.
 Lyophilized cultures can be revived by opening the vials, adding liquid
medium, and transfering the rehydrated culture to a suitable growth
medium.

12. Byophilisation
 The microbial suspension is placed in small vials.
 A thin film is frozen over inside surface of the vial by rotating it in a
mixture of dry ice(solid carbon dioxide), alcohol,or acetone at
temperature of-78°C.
 Immediately connected to a high vaccum- this dries the organism
while still frozen- ampoules are sealed off.
 Cultures can be stored for several years at -40°C.
 To revive microbial cultures, add suitable medium, and after
incubation make further transfers.
 Also for preservation of toxins, enzymes, sera and other biological
material.
 Permits the maintenance of cultures without altering the
characteristics and greatly reduces the danger of contamination.

13. TRANSPORT MEDIA
 Media used for transporting the
samples.
 Special media formulated to preserve a
specimen and minimize bacterial
overgrowth from time of collection to the
time it is received at the laboratory to be
proceed.
 Delicate organisms may not survive the
time taken for transporting the specimen
to the lab or may be overgrown by non-
pathogens.
 Transport media may vary, depending
on the type of organisms in the sample.
 Classification;
on the basis of physical state- semi-
solid & liquid
on the basis of their utility- bacterial
media & viral media.

14. Some important criteria for a Transport
Media
Transport media should fulfil the following criteria:
 Temporary storage of specimens being transported to the laboratory
for cultivation.
 Maintain the viability of all organisms in the specimen without altering
their characteristics.
 Contains only buffers and salt.
 Lack of nitrogen, carbon, and organic growth factors so as to prevent
microbial multiplication.
 Transport media used in the isolation of anaerobes must be free of
molecular oxygen.

15. What are the commonly used Transport
mediums?
 For BACTERIA
 CARY & BLAIR MEDIA
 Semi- solid, white colored.
 Used for Shigella, Salmonella, Vibrio and Campylobacter (usually
Gram- neagtive facultative organisms).
 Containing thioglycolate, phosphate and NaCl.
 Medium supplemented with CaCl2, sodium bisulfite and resazurin for
culture of anaerobes.

17. STUART’S MEDIUM
 Non- nutrient soft agar gel containing a reducing agent to
prevent oxidation.(sodium thioglycolate)
 Used for transporting specimens suspected of having
gonococci.
 Also, used for transporting throat, vaginal, wound and skin
swabs that may contain fastidious organisms.

19. AMIES MEDIUM WITH CHARCOAL
 Useful in the isolation of fastidious organisms.
 Some other pathogens like Campylobacter can also survive in this
medium.
 Charcoal helps eliminate metabolic products of bacterial growth.

20. VENKATARAMAN RAMAKISHNAN (VR)
MEDIUM
 Used to transport faeces from suspected cholera
patients.
SACH’S BUFFERED GLYCEROL
SALINE
used to transport faces from patients suspected to be suffering from
bacillus dysentery.

21. VIRAL TRANSPORT MEDIUM
 Ideal for diagnosis of viral infection.
 Ocular, respiratory and tissue swabs can be submitted.
 Fluid samples such as tracheal wash specimens or peritoneal fluid
submitted as is in sterile vials with several drops of saline added, to
prevent desiccation.
 Special media only used for viruses.

23. ANAEROBIC TRANSPORT MEDIUM
 Mineral salt base semi-solid media with reducing agents such as
sodium thioglycolate and cysteine.
 Designed as a holding medium for maintaining viability of anaerobic
bacteria.
 Resazurin may also be added as a redox indicator to reveal exposure
to oxygen by turning pink.
 Maintains viability of most microorganisms without significant
multiplication and allows for dilution of inhibitors present in clinical
material.
 eg: thioglycolate broth.

25. REFERENCES
 http://preservation stockcultures.html
 http://preserving microbial cultures.html
 http://transport media.html