UNIT III-B: QUANTITATIVE BACTERIOLOGY AND CULTURE PRESERVATION

 Microbial Growth
 Defined as an increase in the number of cells, not cell size.
 Cell synthesis -> polymerization reaction
 Cell growth: assembly of macromolecules, formation of cellular structures
 Bacterial growth: exponential growth

 Generation and Generation Time

 Growth Rate: change in the number of cells or cell mass per unit time.
 Generation: interval for the formation of two cells from one cell.
 Generation time or Doubling time: time required for the formation of two cells from one.

 Binary Fission as Mode of Reproduction

1. Cell elongates and DNA is replicated.
2. Cell wall and plasma membrane begin to divide.
3. Cross-wall forms completely around divided DNA.
4. Cells separate

Bacterial Growth: Binary Fission

 Exponential Growth
 The Microbial Growth Cycle

 Microbial Growth: Growth of Bacterial Cultures

Logarithmic Representation of Bacterial Growth:

We can express the number of cells in a bacterial generation as 2n, where n is the number of
doublings that have occurred.

 Phases of Growth Curve

1. Lag Phase
 Cell synthesizing new components
o e.g., to replenish spent materials
o e.g., to adapt to new medium or other conditions
 Varies in length
o in some cases can be very short or even absent
- nature of medium
- inoculum size and state
-
2. Exponential Phase
 Also called log phase
 Rate of growth and division is constant and maximal
 Population is most uniform in terms of chemical and physical properties during this phase
 3. Stationary Phase
 Closed system population growth eventually ceases, total number of viable cells remains
constant
o active cells stop reproducing or reproductive rate is balanced by death rate

Possible Reasons for Stationary Phase

 Nutrient limitation
 Limited oxygen availability
 Toxic waste accumulation
 Critical population density reached

4. Senescence and Death Phase

 Two alternative hypotheses
o cells are Viable But Not Culturable (VBNC)
 cells alive, but dormant, capable of new growth when conditions are right
 Programmed cell death
o fraction of the population genetically programmed to die (commit suicide)

 Measurement of Growth

1. Total Cell Count

a. Direct Count

i. Counting chamber method (Petroff-hausser chamber)

ii. Coulter Counter method (automated electronic device)
iii. Breed Count (Direct Microscopic Count)

b. Viable Cell Count (Indirect)

- pour plating; spread plating, Miles and Misra (Drop Method), Spiral plate method,
Filtration, Roll tube method (Dairy and food products), Most Probable Number (MPN)

c. Rapid Automated Counts (Indirect)

2. Determination of Mass

a. Direct
- Determination of wet and dry weight
b. Indirect
- Turbidemetric method and Mcfarland standards

3. Measurement by Chemical Analysis

 Direct Count – Counting Chamber Method

PETROFF-HAUSSER CHAMBER

 Direct Count – Breed Count (Direct Microscopic Count)

 A microscopic method of counting the number of cells per mL of milk sample.
 Old technique but cheap and rapid.
 Gives indirect information about the herd from which the milk was drawn.

Formula:
𝑎𝑣𝑒.𝑛𝑜 𝑜𝑓 𝑜𝑟𝑔𝑎𝑛𝑖𝑠𝑚𝑠 𝑝𝑒𝑟 𝑓𝑖𝑒𝑙𝑑 𝑥 𝑛𝑜.𝑜𝑓 𝑚𝑖𝑐𝑟𝑜𝑠𝑐𝑜𝑝𝑖𝑐 𝑓𝑖𝑒𝑙𝑑𝑠 𝑝𝑒𝑟 𝑠𝑞.𝑐𝑚 𝑥 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟
Bacteria/ml = 𝑣𝑜𝑙𝑢𝑚𝑒 𝑢𝑠𝑒𝑑

1
Where: the number of fields in 1 sq.cm = 𝑎𝑟𝑒𝑎 𝑜𝑓 1 𝑚𝑖𝑐𝑟𝑜𝑠𝑐𝑜𝑝𝑖𝑐 𝑓𝑖𝑒𝑙𝑑 𝑖𝑛 𝑠𝑞.𝑐𝑚

*e.g.: under the oil immersion objective (OIO) = 1.77 x 10−4 cm2

 Serial Dilution to Extinction

 Culture Preservation and Maintenance
 Culture Preservation
o to retain the viability and functionality of the stock culture for a long period of time
while maintaining its purity and trait of being “true-to-type”.
 Importance:

1. Ensure the availability, supply, and functionality of microbial strains for future studies.
2. As reference for standardized assays and tests
3. For taxonomic purposes.
4. As valuable stock for biotechnological applications.

 Important Principles for Preservation and Maintenance of Stock Culture

1. Reduction in the required temperature for growth of the organism.
2. Dehydration or desiccation of the growth medium.
3. Limitation of nutrients to the organism.
  Important Things to REMEMBER in Culture Preservation
1. Pure culture and activity growing (in most cases)
2. Sterilize properly all stuff needed.
3. Aseptic technique.
4. Proper labelling (name of the org./code, date, etc.)
5. No. of replicates (more replications in several sizes/location)
6. Proper and secure inventory
7. Protective gear/clothing in handling cultures
8. Proper maintenance of equipment
9. Knowledge about the organism (compatible preservation method)

 Methods in Culturing Microbes

1. Periodic transfer to fresh media 4. Cryopreservation (freezing with liquid

2. Overlying cultures with mineral oil nitrogen)
3. Freeze-drying (lyophilization) 5. Drying
6. Sterile distilled water

 Purity Check of Preserved Microorganisms

 Banking Microbes
 Culture Collections
o Organizations which maintain authentic pure cultures of microorganisms
o Provide ‘type” strains to microbiologists throughout the world
Examples:
o American Type Culture Collection (ATCC), Maryland
o National Collection of Type Cultures (NCTC), London
o Japan Collection of Microorganisms (JCM), Japan
o Philippine National Collection of Microorganisms (PNCM), BIOTECH-UPLB