C3 - The Isolation and Preservation Industrially Important Microorganism

THE ISOLATION, PRESERVATION AND
IMPROVEMENT OF INDUSTRIALLY
IMPORTANT MICRO-ORGANISM
1
Assoc.Prof. Dr. Trịnh Khánh Sơn
Isolation
2
 Isolation involves obtaining either pure or mixed cultures followed by their assessment to
determine which carry out the desired reaction or produce the desired product
Bacterial Isolation and Culture
Assessment: đánh giá

3
 A number of criteria as being important in the choice of organism:

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Points 3,4, and 6 would have to be assessed in detailed tests subsequent to isolation and the
organism most well suited to an economic process chosen on the basis of these results
Amenability: chịu trách nhiệm; genetic manipulation: thao tác gen; ease: dễ dàng
5
Isolation method utilizing selection of the
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desired characteristic
 Enrichment liquid culture (Nuôi cấy làm giàu sinh khối trong môi trường
lỏng)
o Frequently carry out in shake flasks
o Selective force (selective agent) may be re-established by innoculating the
enriched culture into identical fresh medium
o Sub-culturing may be repeated several time before the dominant organism is
isolated by spreading method onto solid medium
7
 Enrichment culture using solidified media (Nuôi cấy làm giàu sinh
khối trên môi trường rắn)
o Solidified media have been used

o Involving selective forces + preliminary diagnostic test (test chẩn đoán
sơ bộ)
8
9
bacteria
clear
zone
10
 Screening methods
o Early screening strategies tended to be empirical (kinh nghiệm), labour
intensive (tay nghề lao động) and had relatively low success rates (trước đây, dựa vào
thực nghiệm và tỉ lệ thành công thấp)
o The number of commercially important compounds isolated

increased, the success rates of such screens decreased further (dùng
các hợp chất đã thương mại để tăng khả năng thành công)
o The evolution of antibiotic screens serves as an excellent

illustration of the development of more precise (chính xác), targeted
system
11
https://www.toku-e.com/Selective-Agents.aspx
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Selective medium
✓ Types are formulated to support the growth of one group of organisms, but inhibit the
growth of another. These media contain antimicrobials, dyes, or alcohol to inhibit the
growth of the organisms not targeted for study. Selective medium types include EMB
agar, Mannitol Salt agar, MacConkey agar, and Phenylethyl Alcohol (PEA) agar.
✓ For example, organisms that can utilize a given sugar are easily screened by making
that sugar the only carbon source in the medium. On the other hand, selective
inhibition of some types of microorganisms can be achieved by adding dyes,
antibiotics, salts or specific inhibitors which affect the metabolism or enzyme systems
of the organisms. For example, media containing potassium tellurite, sodium azide or
thallium acetate (at concentrations of 0.1 -0.5 g/l) will inhibit the growth of Gram-
negative bacteria. Media supplemented with penicillin (5-50 units/ml) or crystal violet (2
mg/l) will inhibit the growth of Gram-positive bacteria. Tellurite agar, therefore, is used
to select for Gram-positive organisms, and nutrient agar supplemented with penicillin
can be used to select for Gram- negative organisms.
https://microbeonline.com/tag/differential-culture-media-used-in-microbiology/
13
Differential medium
✓ Types are those that distinguish microorganisms from one another based
on growth characteristics evident when grown on specific medium types.
Organisms with differing growth characteristics typically show visible
differences in growth when placed on differential media. Examples
include blood agar, Eosin Methylene Blue (EMB) agar, Mannitol Salt agar,
and MacConkey agar.
✓ Differential media are used to differentiate closely related organisms or

groups of organisms. Owing to the presence of certain dyes or chemicals
in the media, the organisms will produce characteristic changes or growth
patterns that are used for identification or differentiation. A variety of
selective and differential media are used in medical, diagnostic and water
pollution laboratories, and in food and dairy laboratories.
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Culture Media for the Isolation of Pathogenic Bacteria from Clinical Specimens
The preservation of industrially important micro-
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organism
 Retains (giữ lại) the desirable characteristics that led to its selection
 The culture used to initiate an industrial fermentation must be viable
and free from contamination
 Microorganism should be cultured to reach the end of log-phase
before preservation
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Storage at reduced temperature

 Cultures grown on agar slopes may be stored in a
refrigerator (5°C) (sub-cultured at approximately weekly or monthly intervals)
 Cultures grown on liquid medium on test-tube and stored
in a freezer ( -40°C, cryoprotective agent) (sub-cultured at
approximately 6-monthly interval)
 The time of subculture may be extended to 01 year if the

slopes (agar slopes) are covered with sterile medicinal grade
mineral oil
Protectants used in the cryopreservation of microorganisms:
17 https://www.ncbi.nlm.nih.gov/pubmed/12818211
 Storage under liquid nitrogen

o The metabolic activities of micro-organisms may be reduced
considerably by storage at the very low temperatures (-150° to -196°C)
o Fungi, bacteriophage, viruses, algae, yeasts, animal and plant cells
and tissue cultures have all been successfully preserved
o Growing a culture to the maximum stationary phase, resuspending the
cells in a cryoprotective agent (tác nhân giúp tế bào chống tổn thương do sự hình
thành tinh thể đá) (such as 10% glycerol) and freezing the suspension in
sealed ampoules before storage under liquid nitrogen
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o Some loss of viability is suffered during the freezing

o And thawing stages (rã đông) but there is virtually no loss during the
storage period.Thus,viability may be predictable even after a period
of many years.
o Although the equipment is expensive the process is economical on
labour.
o However,the method has the major disadvantage that liquid nitrogen
evaporates and must be replenished (bổ sung) regularly
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Storage in a dehydrated form

 Dried cultures (Bảo quản ở môi trường khô)
o Dried soil cultures have been used widely for culture preservation,
particularly for sporulating mycelial organisms
o Then allowed to dry at room temperature for approximately 2 weeks
o Be stored in a dry atmosphere or,preferably, in a refrigerator
o Has been used extensively for the storage of fungi and actinomycetes
after 20-years storage
o Silicagel and porcelain beads are suggested alternatives and detailed
methods are given for these simple, inexpensive techniques
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Lyophylization (đông khô)

 Lyophilization, or freeze-drying, involves the freezing of a culture
followed by its drying under vacuum → sublimation (thăng hoa) of the cell
water.
 Procedure
o Growing the culture to the maximum stationary phase
o Resuspending the cells in a protective medium such as milk, serum or sodium
glutamate
o Transferred to an ampoule → frozen and subjected to a high vacuum until
sublimation is complete
o Ampoules may be stored in a refrigerator and the cells may remain viable for 10
years or more
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 Very convenient for service culture collections

o Once dried, the cultures need no further attention and the storage
equipment (a refrigerator) is cheap and reliable
o Storage at room temperature
o Overall,the technique appears to be second only to liquid nitrogen
storage and even when liquid nitrogenis used makes an excellent
insurance against the possibility of the breakdown of the nitrogen
freezer
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THE ISOLATION, PRESERVATION AND

Bacterial Isolation and Culture

Assessment: đánh giá

 A number of criteria as being important in the choice of organism:

o Solidified media have been used

o The number of commercially important compounds isolated

o The evolution of antibiotic screens serves as an excellent

✓ Differential media are used to differentiate closely related organisms or

Storage at reduced temperature

 The time of subculture may be extended to 01 year if the

 Storage under liquid nitrogen

o Some loss of viability is suffered during the freezing

Storage in a dehydrated form

Lyophylization (đông khô)

 Very convenient for service culture collections

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