Microbial Reproduction and

Growth

Definition of Terms

Growth: Change in total population

Reproduction: that which causes change in population

An increase in cell number is an immediate consequence of cell division.

Modes of Cell Division

Binary fission: 1 cell divides into 2 after developing a transverse septum

Budding: a small bud develops at one end of the cell/ a new cell is formed as an outgrowth from the parent cell (yeast and some bacteria)

Fragmentation: filaments are fragmented into small bacilli

Formation of conidiospores

Calculation

Number of bacteria (N)

N = 1 x 2n

Growth rate (r)

r = n/t = [3.3 (logN – log N0)]/t

Unit: generations/min or h

Calculation

Generation time (g) – time required for 1 cell to divide into 2/time required for the population to double

Generation time (g) = t/nwhere t = elapsed time (hr)

n= number of generations

n= 3.3(logN-logN0)

N = number of cells at the end of the elapsed time

N0= number of cells at the beginning

Unit: min or h/generation

Generation time

1 = parent cell 2 = 1st generation 4 = 2nd generation 8 = 3rd generation 16 = 4th generation

Example

measure culture at 9 a.m.: No = 10,000 cells/ml

measure culture at 3 p.m.: Nf = 100,000 cells/ml

Growth Rate ?Generation time?

Phases of Growth

1. Lag Phase

Bacteria are first introduced into an environment or media

Bacteria are “checking out” their surroundings Cells are very active metabolically # of cells changes very little 1 hour to several days

period of apparent inactivity in which the cells

are adapting to a new environment and

preparing for reproductive growth

2. Log Phase

Rapid cell growth (exponential growth) population doubles every generation microbes are sensitive to adverse conditions

antibiotics anti-microbial agents

3. Stationary Phase

Death rate = rate of reproduction cells begin to encounter environmental stress

lack of nutrients lack of water not enough space metabolic wastes oxygen pH

4. Death Phase

Death rate > rate of reproduction Due to limiting factors in the environment Period in which the cells are dying at an

exponential rate Reasons: continued accumulation of wastes,

loss of cell's ability to detoxify toxins, etc.

Measurement of Bacterial Growth

Serial Dilution and Standard Plate Count Direct Microscopic Count Membrane Filter Count Most Probable Number (MPN) Other methods

Serial Dilution

The process of diluting a sample several times for its microbial count to fall within the countable range (25-250 colonies)

Plate Counts

Pour Plate – adding melted agar to 1.0 ml inoculum

Spread Plate – adding 0.1 ml inoculum on pre-solidified agar and spreading it evenly with a sterile bent glass rod

Pour Plate vs. Spread Plate

Parameters Pour Plate Spread Plate

Size of colonies Small Large

Spreading of colonies Less More

Psychrophiles Good Better

Microaerophiles Better Good

Strict aerobes Good Better

Strict anaerobes Better Not good

Crowding Less More

Pigmentation Good Better

Subculturing Good Better

Reporting Counts

Unit: Colony-forming unit (cfu) per ml or per g Countable range: 25-250 per plate

1. Plates between 25-250

N = C / [ (1xn1)+(0.1x n2)+… ](d) where: N = No. of colonies per ml or g of product

C = Sum of all colonies on plates w/ counts within 25-250

n1 = Number of plates in first dilution counted

n2 = Number of plates in second dilution counted

d = Dilution from which the first count count was obtained

Reporting Counts

2. All plates w/ <10 colonies

N = <25 x 1/d

Where:

d = dilution from which the first count was obtained

3. All plates >250 colonies

N = (plate count nearest 250) x 1/d

Where:

d = dilution from which the the count nearest 250 was obtained

Sample Calculations

1:100 1:1000

A 232 33

B 244 28

= 537/0.022 = 24,409 = 24,000

Condition 1:

Sample Calculations

Colonies EAPC/ml1:100 1:1000

18 2 <2,500

0 0 <2,500

Condition 2:

Sample Calculations

Colonies EAPC/ml1:100 1:1000

TNTC 640 640,000

Condition 3:

Direct Microscopic Count: Petroff-Hausser Counting Chamber

Direct Microscopic Count: Petroff-Hausser Counting Chamber

Unit: Average no. of cell/ml Calculation:

Average no. of cell/ml = 5 squares x 5 x volume of square

Disadvantages: Requires relatively high bacterial densities Cannot distinguish living cells from dead cells

Membrane Filter Method

Sample allowed to pass through a membrane filter

Filter overlayed to a pre-solidified medium

Medium incubated Colonies counted

Most Probable Number (MPN)

Useful particularly when enumerating microorganisms that won’t grow on/in agar media

Used in water quality studies

Growth determined by the production of gas when incubated

95% chance of falling within a particular range of the MPN table

Other Methods

Turbidimetric methods As bacterial cell numbers increase, less light will

reach the photoelectric cell (percentage of transmission)

Metabolic activity Amount of a certain metabolic product is in direct

proportion to the number of bacteria present