Medical Technology

Assessment Program I
BACTERIOLOGY

[TRANS]: BACTERIAL CELL GROWTH

o Once the nutritional requirements are depleted, the
• OUTLINE plateau will start followed by the death phase.
I. Bacterial Cell Growth • E. coli – can complete the growth cycle in about 20
A. Binary Fission minutes.
B. Requirements for Bacterial Cell Growth
i. Nutritional Requirements REQUIREMENTS FOR BACTERIAL CELL GROWTH
ii. Environmental Requirements
II. Nutritional Requirement Nutritional Requirements
A. Oxygen Requirement • Carbon – for synthesis of cellular components
i. Obligate/Strict Aerobes o Sources:
ii. Obligate/ Strict Anaerobe
iii. Facultative Anaerobes
▪ Lithotroph/Autotroph – soure of carbon
iv. Microaerophilic dioxide
B. Carbon Dioxide Requirement ▪ Heterotroph/Organotroph – sources are
i. Capnophilic organic compound (CHON, CHO, Lipids)
ii. For aerobes • Nitrogen – for the synthesis of CHON, RNA, DNA and
iii. For anaerobes ATP
iv. For microaerophilic • Water in the form of moisture
III. Growth
A. Population growth
o The mouth is heavily inhabited by normal floras due
i. Growth rate to moisture.
ii. Generation time • Mineral elements (Mg, Fe, Ca, K)
iii. Growth cycle • Salt requirements – halophilic are salt loving (e.g.,
IV. Measurement of Growth Staph., Vibrio except cholerae and mimicus).
A. Total cell Count o Not all bacteria are not fond of salt.
B. Viable count o In the skin, there are not many pathogenic organisms
C. Turbidimetric Measurements of Cell number
other than those that normal resides in the skin due
to the excretion of sweat and its antibacterial action
BACTERIAL CELL GROWTH which includes a salt content.
• Others – fastidious organisms
BINARY FISSION o Fastidious organisms are those that are hard to
cultivate. Other than the usual requirements needed,
it still needs additional requirements (e.g., moisture
nitrogen, carbon)
o Additional requirements
▪ Haemophilus spp. – X and V factor
o X factor – hemin (heat stable product
release from digestion of Hgb
o V factor – NAD, Coenzyme I (heat labile
produced by some bacteria and yeast.
o Haemophilus grows in the Chocolate Agar
Plate (CAP) because of the blood or
hemoglobin that is already released.
▪ MTB – need increased protein
o Culture media contains whole egg/egg-
based
▪ E.g. Lowenstein Jensen – shows a
gelatin-like appearance

Figure No. 1 Binary Fission
• The bacteria multiplies through the process of binary
fission starting with the DNA replication followed by Cell
elongation then Septum formation in which the bacteria
will eventually be divided into two.
• The time required for a complete growth cycle in bacteria
is highly variable and is dependent on a number of
factors, both nutritional and genetic.
o If the microorganism is fully supplied by the
nutritional requirement it needs, the process of the
growth cycle would be faster. The decline phase
would also be slower as long as there is continuous
supply of the nutritional requirements.
ATENDIDO, LAIZA JANELLE S.
Environmental Requirements
• pH – optimum (best pH for bacterial growth) is neutral or
slightly alkaline
o Acid-loving bacteria – Lactobacillus acidophilus
(pH 3.0)
▪ Media: Tomato juice agar
o Alkali-loving bacteria – (pH 8.0-10.0)
▪ Media: Alkaline peptone water
• Temperature
o Psychrophilic/Cryophilic – optimal temperature for
growth of 15˚C or lower (cold loving), a maximum
growth temperature below 20˚C, and a minimal
temperature for growth at 0˚C or lower.
▪ Ex. Y. enterocolitica, L. monocytogenes
o They require cold enrichment medium
• Mesophilic – require moderate temperature (30˚C-37˚C)
o Most pathogenic bacteria
1
 TRANS: BACTERIAL CELL GROWTH

• Thermophilic – prefer warm temperature (50˚C-60˚C) Table No. 1 Oxygen Relationships of Microorganisms
• Hyperthermophilic – extreme thermophilic (80˚C-110˚C) GROUP
RELATIONSHIP
TO O2
TYPE OF
METABOLISM
EXAMPLE HABITAT
o E.g. Bacillus stearothermophilus Aerobes:
Obligate Required Aerobic Micrococcus Skin,Dust
aerobes respiration luteus (B)
Facultative Not required, but Aerobic, Escherichia coli Mammalian
aerobes growth better anaerobic (B) Large
with O2. respiration, intestine
fermentation
Microaerophilic Required but at Aerobic Spirillum Lake water
aerobes levels lower thn respiration volutans(B)
atmospheric
Anaerobes:
Aerotolerant Not required, Fermentation Streptococcus Upper
anaerobes and growth no pyogenes (B) respiratory
better when O2 tract
present
Obligate Harmful or lethal Fermentation Methanobacterium Sewage
anaerobes or anaerobic formicicum(A) sludge
respiration digestor,
Anoxic lake
sediments

Figure No. 2 Types of Microorganisms classified by optimum

temperature
GROWTH
NUTRITIONAL REQUIREMENT POPULATION GROWTH
OXYGEN REQUIREMENT Growth rate
• Is the change in cell number or cell mass per unit time
Obligate/Strict Aerobes
• During cell division cycle, all the structural components of
• Grows only with the presence of Oxygen (O2) the cell double
• With Catalase and superoxide dismutase enzyme which o Note: In a bacteria, the cells are produce not by cell
convert toxic products of O2 to non-toxic substance division but rather through duplication by means of
o Note: Obligate aerobes can neutralize the waste Binary fission (because bacteria is a prokaryote
product of oxygen because of catalase and organism)
Superoxide dismutase enzyme
▪ The waste product of Oxygen= Carbon dioxide.
Generation time
• Ex. Pseudomonas spp., Neiserria spp., Brucella spp.,
Bordetella spp. & Francisella spp. • The time required for the cell population to double. Also
called Doubling time. (Happens at the end of binary
fission)
Obligate/ Strict anaerobe
• Example of diagram that shows the Doubling time of cells:
• Grows only in the absence of oxygen
• They lack catalase and Superoxide dismutase enzyme Table No. 2 Exponential Growth
• Ex. Bacteroides, Clostridium TIME (h) TOTAL # OF TIME (h) TOTAL # OF
CELLS CELLS
Facultative Anaerobe 0 1 4 256
• Aerobe that can survive even in the absence of Carbon 0.5 2 4.5 512
dioxide 1 4 5 1024
1.5 8 5.5 2048
2 16 6 4096
Aerotolerant Anaerobes
2.5 32 - -
• Anaerobes that can survive in the presence of oxygen 3 64 - -
and have Superoxide dismutase only. 3.5 128 10 1,048,576

Microaerophilic Growth Cycle

• 5-6% oxygen (Usually to bacteria 15-21%oxygen)
• Ex. Campylobacter spp.
CARBON DIOXIDE REQUIREMENT
Capnophilic
• Require 5-10% Carbon dioxide
• Ex. Haemophilus, Neiserria,
Cardiobacterium, Eikenella, Kingella
• For enhancement: candle jar
For Aerobes
• 21% Oxygen, 0.03% Carbon dioxide
For Anaerobes
• Anaerobic jar/chamber: 0% Oxygen, 5%-10% Hydrogen,
5-%10% Carbon dioxide, 80%-90% Nitrogen.
For Microaerophilic
• 5% Oxygen, 10% Carbon dioxide, 85% Nitrogen
1. Lag phase
o It is the period where the individual bacteria are
maturing and not yet able to dublicate. During the lag
phase of the bacterial growth cycle, synthesis of
RNA, enzymes and other molecules occurs. So in
this phase the microorganism is dormant.
2. Log/Exponential Phase
Actinobacillus,
o The cell number doubles within a fixed time period.
o Where bacteria duplicates.
o If growth is not limited, doubling will continue at a
constant rate so both the number of cells and the rate
of population increase doubles with each
consecutive time period.
o Most active metabolically, most susceptible to
antimicrobial agents.
3. Plateau/Stationary Phase
o The growth rate slows as a results of nutrition
depletion, development of unfavorable pH and
accumulation of toxic products.
o There is no net increase or decrease in cell number.
o The rate of bacterial growth is equal to the rate of
bacterial death.
4. Decline / Degaradtion / Death Phase

ATENDIDO, LAIZA JANELLE S. 2

 TRANS: BACTERIAL CELL GROWTH

o Bacteria run out of nutrients and die.

o In some cases death is accompanied by cell lysis.

Figure No. 3 Growth Cycle

Figure No. 5 Dilution method

MEASUREMENT OF GROWTH
• Total cell count
o Direct microscopic count TURBIDIMETRIC MEASUREMENTS OF CELL
▪ Samples dried on slides NUMBER
▪ Samples in liquid- “turbidity” as a sign of • Turbidity of the cell suspension is measured with:
bacterial growth o Photometer
• Viable count o Spectrophotometer
• Turbidimetric measurements of cell number ▪ Both measures scattered light.
▪ The more cells present, the more light scattered
TOTAL CELL COUNT: LIMITATIONS OF DIRECT and hence the more turbid the suspension.
MICROSCOPIC COUNTING
REFERENCES
• Dead cells are not distinguish from living cells.
• Small cells are difficult to see. Notes from the discussion by Mr. Edison Ramos, RMT, MPH,
• Precision is difficult to achieve. MSMT
• A phase contrast microscope is required when sample
is unstained. National University powerpoint presentation: Bacterial Cell Growth
• No suitable for cell suspensions of low density.

VIABLE COUNT
• Viable – one that is able to divide and form offspring.
• Colony count/Plate count – done by determining the
number of cells in the sample capable of forming colonies
on a suitable agar medium.

Methods in Viable counting

• Spread plate method
o Sample is pipetted onto the surface of the ready-
made agar.
o Sample is spread evenly over surface of agar using
sterile glass spreader.
• Pour-plate method
o Sample is pippeted first into the sterile plate.
o Sterile medium is then added.
• Dilutions

Figure No. 4 Spread-plate and Pour-plate methods

ATENDIDO, LAIZA JANELLE S. 3