Microbial Growth

Microbial Nutrition & Microbial Nutrition
Microbial Growth MICROORGANISMS REQUIRE A SUITE OF

NUTRIENTS AS FOODSTUFFS
TO P R O D U C E N E W M I C R O B I A L C E L L S .
PREPARED BY: FLORENCE JHUN F. ALMADIN
Microbial Nutrition Microbial Nutrition

Cultivation:
Microorganisms vary in their requirement for nutrients
-Increasing the population of microorganisms by providing
their nutritional and physical requirements
Nutrients: Knowledge of the principles of microbial nutrition
-Extracellular substances which provide the cell with
materials for building protoplasm and for energy Necessary for their successful cultivation in the laboratory
generation
PREPARED BY: FLORENCE JHUN F. ALMADIN PREPARED BY: FLORENCE JHUN F. ALMADIN

Not all nutrients are required in the same amounts
Nutrients: constructed from chemical elements
Macronutrients = needed in relatively larger quantities
Examples: C, O, N, H (predominantly required)

P, S, K, Na, Mg, Ca, etc. Data are for E. coli (~comparable data for other
microorganisms); cited in Mardigan et al., 2015)

Micronutrients = needed in minute/trace quantities To cultivate microorganisms in the laboratory use
= include metals and growth factors (vitamins, amino acids, of:
purines & pyrimidines)
Culture Media = any nutrients material
prepared/used for the growth and cultivation
Example of Metals: B, Co, Cu, Fe, Mn,
of microorganisms in the laboratory
Mo, Ni, Se, W, V, Zn, etc.,

Uses of Culture Media: TYPES OF CULTURE MEDIA:
1. According to Physical State or Consistency
1. For the growth and maintenance of microbial culture ◦ A. liquid (or Broth): with no solidifying agent (e.g. Agar,
gelatin, carrageenan)
2. To favor the production of particular compounds
: e.g. Nutrient Broth
3. To study microbial action on some constituents of the
medium

TYPES OF CULTURE MEDIA:
TYPES OF CULTURE MEDIA:
1. According to Physical State or
Consistency 1. According to Physical State or Consistency NA
B. Semi-solid: with 0.1-0.5% solidifying C. Solid: with 1.5-2.0% solidifying agent
agent e.g. Nutrient Agar (NA), Blood Agar (BA)
e.g. Sulfide Indole Motility (SIM)
Medium
BA
AGAR Types of Culture Media:
ØComplex polysaccharide 2. According to Chemical Composition
ØUsed as solidifying agent for culture media in petri
plates, slants, and deeps
A. Synthetic: all components are chemically defined
ØGenerally not metabolized by microbes e.g. Glucose Inorganic Salts Phosphate for E. coli
ØLiquefies at 100 c (see table 6.2)
ØSolidifies at 40 c B. Complex: not all components are chemically defined
e.g. NA, potato infusion, yeast extract, beef extract
(see table 6.4)
A. Synthetic culture media: B. Complex culture media:

Example of Ingredients Added in Complex Media Types of Culture Media:
Beef extract: concentrate of hot aqueous infusion of fresh 3. According to Principal Purpose, Function
beef or Application
Peptone: Spray dried hydrolysate of various proteins A. General Purpose: can support most or
almost all types of species
Yeast extract: Spray dried water soluble autolyzed yeast
cells e.g NA
Tryptic Soy Agar (TSA)
Brain Heart Infusion (BHI) Agar
Microbial Nutrition EMB Agar
Types of Culture Media: ØOnly gram-negative bacteria grow on EMB
3. According to Principal Purpose, Function or Application agar.
B. Differential: can distinguish visually one type of bacterium ØGram-positive bacteria are inhibited by the
from another; with special reagents like pH indicators, dyes eosin and methylene blue added to the
dyes, etc. agar.
E.g. Eosin Methylene Blue Agar (EMBA) ØBased on its rate of lactose fermentation, E.
Mannitol Salt Agar (MSA) coli produces dark, blue-black colonies with a
metallic green sheen on EMB agar.
Microbial Nutrition MSA: Selective and Differential

Types of Culture Media:
3. According to Principal Purpose, Function or
Application
C. Selective: allows the growth of a specific type
of microorganism only; inhibits others; contain
selective agents (Salts, dyes, antibiotics, etc.,)
e.g. Bacillus cereus Agar (contains polymyxin,
Na pyruvate & lecithin)
Microbial Nutrition Blood Agar

Alpha-hemolysis = incomplete lysis of (RBCs – appears green)
3. According to Principal Purpose, Function or Application
Beta-hemolysis = total lysis of RBCs (complete clearing)
D. Enrichment: used to increase the number of microorganisms
Gamma-hemolysis = no hemolysis
with unusual physiological characteristics; contains special
nutrient(s) for the microbe of interest (e.g. cellulose, petroleum,
blood)
e.g. Cellulose Agar (for cellulotic microorganisms)
Petroleum Broth (for petroleum-utilizers)
Blood Agar (for hemolytic bacteria; also differential)
Microbial Nutrition Fermentation Medium Assay
3. According to Principal Purpose, Function or Application
Assay: used for assay of vitamins, amino acids, antibiotics, etc.; of
prescribed composition; may be used for the qualitative or quantitative
production of such a compound by a microorganism
E.g. Fermentation media,
Triple Sugar Iron (TSI) Agar
Media for Antibiotics Sensitivity Testing
Vitamin B12 Assay Medium
Triple Sugar Iron Test Antibiotic Sensitivity Testing

Preparation of Culture Media
Preparation of Culture Media: General Steps Sample Problem 1
1. Weighing of hydrated media or media ingredients Given the following composition of Medium MCB:
2. Dissolving of components in the solvent Yeast extract – 1.5 g MgSo4 – 0.5 g
3. Checking/adjustment of pH (optional) – prior to agar addition Sucrose – 7.0 g Agar – 15.0 g
4. Dispensing into glassware/containers Distilled water – 1000 ml
5. Capping or plugging with cotton A. Determine the total volume of Medium MCB needed if you are to prepare for 10 plates
(regular size), 15 agar slants (big tube) and 20 agar stabs or agar deeps (big tube)
6. Sterilization – usually at 15 psi for 15 minutes Assumptions: per plate = 15-20 ml; per agar slant (big tube) = 6-8 ml
per agar stab = 10-12 ml
B. Compute for the amount of each component needed
Preparation of Culture Media Preparation of Culture Media
Solution Sample Problem 1 Solution Sample Problem 1
10 plates x 20 ml/plate = 200 ml B. Using Ratio and Proportion:
For YE 1.5 g = xg = 0.99 g
15 agar slants x 8 ml/slants = 200 ml 1000 ml 660 ml
20 agar stabs x 10 ml/stabs = 200 ml For Suc 7.0 g = xg

660 ml
= 4.62 g
1000 ml
Exact volume of medium MCB ` = 600 ml + 60 ml For MgSO4 0.5 g = xg = 0.33 g
1000 ml 660 ml
Total Volume to be prepared = 660 ml For Agar 1.5 g = xg = 9.90 g

1000 ml 660 ml
For dH2O 660 ml

Microbial Nutrition Solution Sample Problem 2
Preparation of Culture Media Using ratio and proportion:
For Tryp 0.5 g = Xg = 2.00 g
Sample Problem 2 1000 ml 400 ml
Medium XYZ has the following composition: For Dextrose 15.0 g = Xg = 6.00 g
1000 ml 400 ml
Tryptone – 0.5% Yeast Extract – 1.0 g/L For YE 1.0 g = Xg = 0.40 g
Dextrose – 15.0 g/L Agar - 1.6 % 1000 ml 400 ml
For Agar 1.5 g = Xg = 6.40 g

If you need a total volume of 400 ml of Medium XYZ, how much 1000 ml 400 ml
of each of the medium components should you weigh/measure? For dH2O 400 ml

Microbial Nutrition Solution Sample Problem 3
Preparation of Culture Media Needed: 100 ml of 2M Na2CO3; available is Na2CO33H2O
Sample Problem 3
One method: Compute first the amount needed for Na2CO3.
A medium formulation to be supplemented with 5ml/L of a 2M Based on formula: M = moles = wt / Mol. Wt.
solution of Na2Co2 L L
How will you prepare 100 ml of 2M Na2CO3 given that what is and the Mol. Wt. of Na2CO3 = 106 g/mol
available is Na2CO3 + 3H2O? xg Na2CO3 = 21.2 g
Atomic Mass (g/mol) : Na = 23.0
C = 12.0
O = 16.0
H = 1.0
Solution Sample Problem 3 Microbial Nutrition
Since what is available is Na2CO33H2O : Preparation of Culture Media
Sample Problem 4
Mol. Wt. Na2CO3 = 106.0 = 21.2 g
Mol. Wt. Na2CO33H2O 160.0 xg IF 2.0 ML OF A 10% SOLUTION OF PHENOL WAS TRANSFERRED
TO 8.0 ML OF NUTRIENT Broth (NB) in a tube, give the final
concentration of phenol in NB tube.
◦ xg = 32.0 g of Na2CO33H2O in 100 ml of dH2O

Solution Sample Problem 4 Acknowledgment:
Using : C1V1 = C2V2 CRUZ, WILMA T., AND WAJE, CARMELA C., Lecture notes in General Microbiology Division, IBS,
UPLB
MENDOZA, BERNADETTE C., Lecture notes in Microbial Nutrition and Control of Microbial
Growth, Control of Microbial Growth; Module 1 : Training Basic Microbiology, Training capacity
building of tertiary educators in the teaching of selected microbiology courses for the members
of the microbiology consortia in Philippines, IBS, UPLB
(10.0%) (2.0ml) = (X%) (10.0 ml)
X% = 2.0%
Microbial Growth
- Orderly increase of all the chemical constituents of an organism

Ø cellular constituents
Ø cellular structure
UNIT 4. - growth without cell division
MICROBIAL
GROWTH - increase in size and weight of the cell
Ø For most microorganisms: growth followed by cell division

BIOL 106 and increase in cell number
GENERAL MICROBIOLOGY
Ø Increase in total mass – not necessarily a reflection of
growth
Microbial Growth Microbial Growth
Stages/Phase of
Microbial Growth:
ØOrganisms that divide by • Lag Phase

BINARY FISSION
• Log or exponential
– not possible to distinguish
growth phase
resulting cells from the mother
cell • Stationary phase
• Death or
Logarithmic decline
phase
1. Lag phase 3. Stationary phase

• no immediate increase in cell number or mass Varies considerably in length with - no net increase or decrease in cell number (cryptic growth)
• cells are dormant but physiologically active the condition of the microorganisms
and the nature of the medium
- slow metabolic activity
• synthesis of new protoplasmic constituents - cells enter this phase due to the following reasons:
• synthesis of new enzymes
• adaptation phase
1. nutrient limitation
2. accumulation of toxic products
2. Exponential or log phase - microorganisms are growing and dividing at the maximal rate - cells are smaller than those in the log phase and do not have constant
possible → dependent on the following: composition!
• genetic potential of the organism
• nature of the medium
• conditions under which they are growing 4. Death phase
Cells nearly uniform in chemical composition and physiological characteristics! - death rate > growth rate
- Rate of growth is constant - death is logarithmic but, in most cases, slower than that of exponential
- Microorganisms divide and double in number at regular intervals growth
Typical Bacterial Growth Curve Typical Bacterial Growth Curve
Environmental Factors That Influence

Microbial Growth
• Prokaryotes inhabit nearly all environments Environmental

• Some live in comfortable habitats favored by humans Factors That
• Some live in harsh environments Influence
• Termed extremophiles; most are Archaea Microbial
• Major conditions that influence growth Growth
• Temperature
• Atmosphere (Oxygen, Carbon dioxide)
• pH
• Water availability
4.4. Temperature Requirements Temperature Requirements

• Each species has well-defined temperature range • Proteins of thermophiles resist denaturing
• Optimum growth usually close to upper end of range • Thermostability comes from amino acid sequence
• Psychrophiles: –5° to 15°C • Number and position of bonds which determine structure
• Found in Arctic and Antarctic regions
• Temperature and food preservation
• Psychrotrophs: 15° to 30°C
• Refrigeration (~4°C) slows spoilage by limiting growth of
• Important in food spoilage
otherwise fast-growing mesophiles
• Mesophiles: 25° to 45°C • Psychrophiles, psychrotrophs can still grow, but slowly
• Pathogens 35° to 40°C
• Freezing preserves food; not effective at killing microbes
• Thermophiles: 45° to 70°C
• Common in hot springs • Temperature and disease
• Hyperthermophiles: 70° to 110°C • Temperature of different parts of human body differs
• Usually members of Archaea • Some microbes cause disease in certain parts
• Found in hydrothermal vents • For example Hansen’s disease (leprosy) in coolest regions (ears, hands,
feet, fingers) due to preference of M. leprae
Oxygen Requirements Oxygen Requirements
• Shake tube • Reactive oxygen species
• Boil nutrient agar to drive off O2; cool to just above solidifying • Using O2 in aerobic respiration produces harmful reactive oxygen
temperature; inoculate; gently swirl species (ROS) as by-products
• Growth demonstrates organism’s O2 requirements • Includes superoxide (O2–) and hydrogen peroxide
• Damaging to cellular components
• Cells must have mechanisms to protect
• Obligate anaerobes typically do not
• Almost all organisms growing in presence of oxygen produce
enzyme superoxide dismutase
• Inactivates superoxide by converting to O2 and H2O2
• Almost all also produce catalase
• Convert H2O2 to O2 and H2O
• Exception is aerotolerant anaerobes; makes for useful test
pH Water Availability
• Bacteria survive a range of pH; have optimum
• Most maintain constant internal pH, typically near neutral • All microorganisms require water for growth
• Pump out protons if in acidic environment • Dissolved salts, sugars make water unavailable to cell
• Bring in protons if in alkaline environment • If solute concentration is higher outside of cell, water diffuses out
(osmosis)
• Most microbes are neutrophiles
• Salt, sugar used to preserve food
• Range of pH 5 to 8; optimum near pH 7
• Food can be preserved by increasing acidity • Some microbes withstand or even require high salt
• Helicobacter pylori grows in stomach; produces urease to split urea • Halotolerant: withstand up to 10% (Staphylococcus)
into CO2 and ammonia to decrease acidity of surroundings • Halophiles: require high
• Acidophiles grow optimally at pH below 5.5 salt concentrations
• Picrophilus oshimae (hot spring) has optimum pH of less • Marine bacteria ~3%
than 1! (thermoacidophilic) • Extreme halophiles ≥ 9%
(Dead Sea, Utah’s salt flats)
• Alkaliphiles grow optimally at pH above 8.5
Measuring Microbial Growth Measuring Microbial Growth
Ways to measure microbial growth:

1. Cell count
2. Cell mass
3. Cell activity (Enzymes and Biochemical activities)
Methods employed in inoculating

Petri plates from the serial
dilutions.
1. Pour Plate
2. Spread Plate
Lactic Acid Bacteria – Spread Plate (0.1 ml volume plated) Lactic Acid Bacteria
Food Sample 10-3 10-4 10-5 Food Sample 10-3 10-4 10-5
Yoghurt 400, 347 255, 234 34, 25 Yoghurt 400, 347 255, 234 34, 25
Pickle 259, 291 106, 103 20, 23 Pickle 259, 291 106, 103 20, 23
Note: Valid counts: 30 – 300 (Bacteria and non spreaders), 10-150 (Yeast, Note: Valid counts: 25 – 250 (Bacteria and non spreaders), 10-150 (Yeast,
actinomycetes and molds) actinomycetes and molds)
Yoghurt : Yoghurt :
CFU/ml = 234+ 34+ 25 5 CFU/ml = 234+ 34+ 25 5 CFU/ml = 2.4 X 107

((1 x 1) + (0.1 x 2)) (10-4 ) ( 0.1 ml) ((1 x 1) + (0.1 x 2)) (10-4 ) ( 0.1 ml)
Pickle: Pickle:
CFU/ml = 104.5 x 1x104 CFU/ml = 104.5 x 1x104 CFU/ml = 1.04 X 107

0.1 ml 0.1 ml

Measuring Microbial Growth Standard Curve
• The study of bacterial growth curves is important when aiming to utilize or inoculate known numbers of
the bacterial isolate, for example to enhance plant growth, increase biodegradation of toxic organics, or
produce antibiotics or other natural products at an industrial scale.
• Knowledge of bacterial growth kinetics and bacterial numbers in a culture medium is important from both
a research and commercial point of view.
Standard Curve Standard Curve
16-24 hrs bacterial Control Tubes Stock Suspension (ml) Diluent(ml) Dilution
Plate Count (CFU/ml)
OD Reading (600) stock suspension 1 0.2 4.8 4:100
2 0.3 4.7 6:100
3 0.4 4.6 8:100
4 0.5 4.5 10:100
5 0.6 4.4 12:100
6 0.7 4.3 14:100
7 0.8 4.2 16:100
8 0.9 4.1 18:100
9 1.0 4.0 20:100
Get the cell population

Beer Lambert’s Law Linear equation (CFU/ml) Then get the cells/ml at each particular dilution (control) tubes
Ø is a linear relationship between the absorbance and
y = mx + b
the concentration cells/ml for particular dilution = cfu/ml of stock suspension used X vol of stock used
total volume
Control Tubes OD Reading Cells/ml at a OD Reading

Control Tubes
16-24 hrs bacterial 16-24 hrs bacterial particular dilution
stock suspension
1 stock suspension 1 13600000 0.032
2
2 20400000 0.047
3
3 27200000 0.056
4
4 34000000 0.070
5
5 40800000 0.080
6
6 47600000 0.091
7
7 54400000 0.100
8
Spectrophotometer 8 61200000 0.107
9
9 68000000 0.119
Plot in Excel (Linear Equation)
Can vary depending on the

Cells/ml at a OD Cells/ml at a OD equation of the line
Control particular Reading
particular Reading Time
Tubes dilution (x)
dilution
y = 2E-09x + 0.0144
1 13600000 0.032 1 0.032
2 20400000 0.047 2 0.047
3 27200000 0.056 3 0.056
4 34000000 0.070 4 0.070 x = y – 0.0144
5 40800000 0.080 5 0.080 2E-09
6 47600000 0.091 6 0.091
7 54400000 0.100 7 0.100
8 61200000 0.107 8 0.107
where y, is your OD value
9 68000000 0.119 y = 2E-09x + 0.0144 9 0.119 and x, cells/ml
R² = 0.9943
Growth Curve Growth Curve
Cells/ml at a OD Cells/ml at a OD
Time particular Reading Time (hrs) particular Reading
dilution (x) dilution (x)
1 8800000 0.032 x = y – 0.0144 1 8800000 0.032
2 16300000 0.047 2E-09 2 16300000 0.047
3 20800000 0.056 3 20800000 0.056
4 27800000 0.070 4 27800000 0.070
where y, is your OD value
5 32800000 0.080 5 32800000 0.080
6 38300000 0.091
and x, cells/ml 6 38300000 0.091
7 42800000 0.100 7 42800000 0.100
8 46300000 0.107 8 46300000 0.107
9 52300000 0.119 9 52300000 0.119
Quantitative aspects of Microbial Growth Quantitative aspects of Microbial Growth
v Increase in cell number that occurs in an exponentially

Generation: growing bacterial culture is a geometric progression of the
number 2!
-growth by binary fission
Generation rate: Nf = N 0 x 2n
-change pf bacterial population Where:
over a period of time (cells/time). Nf = final cell number
N0 = initial cell number
Generation Time: n = number of
-the time it takes for a population generations
of bacteria to double in number.
Quantitative aspects of Microbial Growth Quantitative aspects of Microbial Growth

► to solve for n: Nf = N 0 x 2n
Where:
n = log Nf – log N0 = log Nf – log N0
log 2 0.301 Nf = final cell number
N0 = initial cell number
v generation time (g) = time required for the population to double n = number of
generations
g = t/n where: t = number of hours/minutes
of exponential growth
In a short test tube fermentation experiment, we are looking for the
final cell population of an E. coli with an initial cell population of
236 cells. The experiment will be terminated after 10 generations.
Ø graphical estimation of g Note that the doubling time of E. coli is 20 minutes.
by plotting data on
semilogarithmic graphs
Nf = 236 X 210
= 236 X 1024
= 241664 cells
= 2.4 X 105
Daghang Salamat!!
Chapter 4
MICROBIAL GROWTH
(Microbial Growth)
Leila A. Ombat, PhD See separate PowerPoint slides for all figures and tables pre-
inserted into PowerPoint without notes.
CSU-CMNS-Department of Biology
Copyright © McGraw-Hill Education. Permission required for reproduction or display. 1 Copyright © McGraw-Hill Education. Permission required for reproduction or display. 2
PART 1 - SUBTOPICs
1. A glimpse of history.
2. Introduction
3. Principles of bacterial growth
MICROBIAL GROWTH 4. Prokaryotic growth in nature
5. Biofilms
(Part 1)
6. Interaction in mixed microbial communities
7. Obtaining a pure culture
Leila A. Ombat, PhD 8. Growing microorganism in solid medium
CSU-CMNS-Department of Biology 9. Prokaryotic growth in laboratory conditions
10. Growth curve
11. Colony growth
12. Continuous culture
A Glimpse of History
PART 2- SUBTOPICs
13. Environmental factors that influence microbial growth § German physician Robert Koch (1843–1910)
- temperature • Studied disease-causing bacteria; Nobel Prize in 1905
- oxygen • Developed methods of cultivating bacteria
- pH • Worked on methods of solid media to allow single
- water availability bacteria to grow and form colonies
• Tried potatoes, but nutrients limiting for many bacteria
14. Nutritional factors that influence microbial growth • Solidifying liquid nutrient media
15. Culturing microbes in the laboratory with gelatin helped
- providing appropriate atmospheric conditions
• Limitations: melting temperature,
- enrichment cultures digestible
16. Methods to detect and measure microbial growth
• In 1882, Fannie Hess, wife of
associate, suggested agar she
used to harden jelly
Copyright © McGraw-Hill Education. Permission required for reproduction or display. 5

Introduction The Microbial Growth
§ Prokaryotes can be found growing in severe

Learning Outcomes
conditions
• Ocean depths, volcanic vents, polar regions all harbor
thriving prokaryotic species
1. Define bacterial growth including binary fission.
• Many scientists believe that if life exists on other 2. Compare the phases of microbial growth, and
planets, it may resemble these microbes describe their relation to generation time
§ Individual species have limited set of conditions 3. Describe the formation of biofilms and their
• Also require appropriate nutrients potential for causing infection.
§ Important to grow microbes in culture 4. Define colony.
• Medical significance 5. Describe how pure cultures can be isolated by
• Nutritional, industrial uses using the streak plate method.
4.1. Principles of Bacterial Growth 4.1. Principles of Bacterial Growth
§ Prokaryotic cells divide by § Growth can be calculated

binary fission • Nt = N0 x 2n
• One cell divides into two, two into • Nt = number of cells in population at time t
four, 4à8, 8à16, etc…
• Exponential growth: population • N0 = initial number of cells
doubles each division • n = number of generations at that point
• Generation time is time it takes for • Example: pathogen in potato salad at picnic in sun
the population to double • Assume 10 cells with 20 minute generation time
• Varies among species
• N0 = 10 cells in original population
• Environmental conditions
• n = 12 (3 divisions per hour for 4 hours)
• Exponential growth has important
consequences • Nt = N0 x 2n = 10 x 212
• 10 cells of food-borne pathogen in • Nt = 10 x 4,096
potato salad at picnic can become • Nt = 40,960 cells of pathogen in 4 hours!
40,000 cells in 4 hours
4.1. Principles of Bacterial Growth 4.2. Prokaryotic Growth in Nature
§ The power of exponential growth § Microorganisms historically studied in laboratory

• Rapid generation time with optimal conditions can § But dynamic, complex conditions in nature have
yield huge populations quickly profound effect on microbial growth, behavior
• Remember that generation time depends on species • Cells sense changes, adjust to surroundings
and growth conditions
• Synthesize compounds useful for growth
• Most live in polysaccharide-
encased communities
termed biofilms
• Cause slipperiness of rocks
in stream bed, slimy “gunk”
in sink drains, scum in toilet
bowls, dental plaque
4.2. Biofilms 4.2. Biofilms
§ Formation of biofilm § Biofilms have characteristic architectureChannels

through which nutrients and wastes pass
• Cells communicate by synthesizing chemical signals
§ Biofilms have important implications
• Dental plaque leads to tooth decay, gum disease
• Most infections (e.g., ear infections, cystic fibrosis)
• Industrial concerns: accumulations in pipes, drains
• Biofilm structure shields microbes growing within
• May be hundreds of times more resistant to disinfectants
• Biofilms can also be helpful
• Bioremediation, wastewater treatment
4.2. Interactions of Mixed Microbial Communities 4.3. Obtaining a Pure Culture
§ Prokaryotes regularly grow in close association § Pure culture defined as population of cells derived
• Many different species from a single cell
• Interactions can be cooperative • Allows study of single species
• Can foster growth of species otherwise unable to survive § Pure culture obtained using aseptic technique
– Strict anaerobes can grow in mouth if others consume O2
• Minimizes potential contamination
– Metabolic waste of one can serve as nutrient for other
• Interactions often competitive § Cells grown on culture medium
• Some synthesize toxic compounds to inhibit competitors • Contains nutrients dissolved in water
• Some Gram-negative bacteria use the needle-like • Can be broth (liquid) or solid gel
structure called a type VI secretion system to inject toxic
compounds directly into competing bacteria.
4.3. Growing Microorganisms on Solid Medium 4.3. Growing Microorganisms on Solid Medium
§ Need culture medium, container, aseptic conditions, § Streak-plate method

method to separate individual cells • Simplest, most commonly used method for isolating
• With correct conditions, single cell will multiply
• Spreads out cells to separate
• Form visible colony (~1 million cells easily visible)
• Agar used to solidify
• Obtain single cells so that individual colonies can form
• Not destroyed by high temperatures and can be sterilized
• Liquifies above 95°C
• Solidifies below 45°C
• Few microbes can degrade
• Growth in Petri dish
• Allow air
• Excludes contaminants
• Culture medium in
a Petri dish called a plate
4.3. Growing Microorganisms on Solid Medium 4.3. Prokaryotic Growth in Laboratory Conditions
§ Maintaining stock cultures § Prokaryotes grown on agar plates or in tubes or

• Streak-plate method yields pure cultures flasks of broth
• Can be maintained as stock culture • Closed systems
• Often stored in refrigerator as agar slant • Nutrients not renewed; wastes not removed
• Cells can be frozen at –70°C for long-term storage • Termed batch cultures
• Mixed with glycerol to prevent ice crystal formation • Yields characteristic growth curve
• Can be freeze-dried
4.3. The Growth Curve 4.3. The Growth Curve
§ Growth curve characterized by five stages § Lag phase

• Number of cells does not increase
• Begin synthesizing enzymes required for growth
• Delay depends on conditions
§ Exponential (log) phase
• Cells divide at constant rate
• Generation time measured
• Most sensitive to antibiotics
• Production of primary
metabolites
• Important commercially
• Secondary metabolite production occurs as nutrients are
depleted and wastes accumulate
4.3. The Growth Curve 4.3. Colony Growth
§ Stationary phase § Colonies and liquid cultures share similarities

• Nutrient levels too low to sustain growth § Important differences based on location
• Total numbers remain constant • Position of single cell determines its environment
• Some die, release contents; others grow • Edge of colony has O2, nutrients
§ Death phase • Center of colony has depleted O2, nutrients
• Total number of viable cells decrease • Accumulation of potentially toxic wastes including acids
• Cells die at constant rate • Colony may range from exponential growth at edges to
• Exponential, but usually much slower than cell growth death phase in center
§ Phase of prolonged decline
• Some fraction may survive
• Adapted to tolerate worsened conditions
Morphology of bacterial colony Fungal Colony
Bacterial colonies 4.4. Continuous Culture
§ Open system required to maintain continuous

growth
• Termed continuous culture
• Nutrients added, wastes removed continuously
§ Chemostats can maintain continuous growth
• Continually drips fresh medium into culture in chamber
• Equivalent volume removed
• Contains cells, wastes, spent medium
• Nutrient content and speed of addition can be controlled
• Achieve constant growth rate and cell density
• Produces relatively uniform population for study
Up Next
MICROBIAL GROWTH MICROBIAL GROWTH

(Part 2) (Part 2)
Leila A. Ombat, PhD


Review on Streak Plate Method Part 2 Microbial Growth Subtopics
13. Environmental factors that influence microbial growth

- temperature
- oxygen
- pH
- water availability
14. Nutritional factors that influence microbial growth

15. Culturing microbes in the laboratory
- providing appropriate atmospheric conditions
- enrichment cultures
16. Methods to detect and measure microbial growth
Environmental Factors
LEARNING OUTCOMES
1. Classify microbes into five groups on the basis of preferred

REQUIREMENTS FOR GROWTH temperature range.
2. Identify how and why pH of cultures is controlled.
3. Explain the importance of osmotic pressure to microbial growth.
Environmental Factors
4. Name a use for each of the four elements (carbon, nitrogen, sulfur
Nutritional Factors and phosphorus) needed in large amounts for microbial growth.
5. Explain how microbes are classified on the basis of oxygen
requirements.
6. Identify ways in which aerobes avoid damage by toxic forms of
oxygen.
4.4. Environmental Factors That Influence 4.4. Environmental Factors That Influence
§ Prokaryotes inhabit nearly all environments
• Some live in comfortable habitats favored by humans
• Some live in harsh environments
• Termed extremophiles; most are Archaea
§ Major conditions that influence growth
• Temperature
• Atmosphere (Oxygen, Carbon dioxide)
• pH
• Water availability
4.4. Temperature Requirements 4.4. Temperature Requirements
§ Each species has well-defined temperature range § Proteins of thermophiles resist denaturing
• Optimum growth usually close to upper end of range • Thermostability comes from amino acid sequence
• Psychrophiles: –5° to 15°C • Number and position of bonds which determine structure
• Found in Arctic and Antarctic regions § Temperature and food preservation
• Psychrotrophs: 15° to 30°C • Refrigeration (~4°C) slows spoilage by limiting growth of
• Important in food spoilage otherwise fast-growing mesophiles
• Mesophiles: 25° to 45°C • Psychrophiles, psychrotrophs can still grow, but slowly
• Pathogens 35° to 40°C • Freezing preserves food; not effective at killing microbes
• Thermophiles: 45° to 70°C
§ Temperature and disease
• Common in hot springs
• Temperature of different parts of human body differs
• Hyperthermophiles: 70° to 110°C
• Some microbes cause disease in certain parts
• Usually members of Archaea
• For example Hansen’s disease (leprosy) in coolest regions
• Found in hydrothermal vents (ears, hands, feet, fingers) due to preference of M. leprae
4.4. Oxygen Requirements 4.4. Oxygen Requirements
§ Shake tube § Reactive oxygen species

• Boil nutrient agar to drive off O2; cool to just above solidifying • Using O2 in aerobic respiration produces harmful reactive
temperature; inoculate; gently swirl oxygen species (ROS) as by-products
• Growth demonstrates organism’s O2 requirements • Includes superoxide (O2–) and hydrogen peroxide
• Damaging to cellular components
• Cells must have mechanisms to protect
• Obligate anaerobes typically do not
• Almost all organisms growing in presence of oxygen
produce enzyme superoxide dismutase
• Inactivates superoxide by converting to O2 and H2O2
• Almost all also produce catalase
• Convert H2O2 to O2 and H2O
• Exception is aerotolerant anaerobes; makes for useful test
4.4. pH 4.4. Water Availability

§ Bacteria survive a range of pH; have optimum
• Most maintain constant internal pH, typically near neutral § All microorganisms require water for growth
• Pump out protons if in acidic environment • Dissolved salts, sugars make water unavailable to cell
• Bring in protons if in alkaline environment • If solute concentration is higher outside of cell, water
diffuses out (osmosis)
• Most microbes are neutrophiles
• Salt, sugar used to preserve food
• Range of pH 5 to 8; optimum near pH 7
• Some microbes withstand or even require high salt
• Food can be preserved by increasing acidity
• Helicobacter pylori grows in stomach; produces urease to • Halotolerant: withstand up to 10% (Staphylococcus)
split urea into CO2 and ammonia to decrease acidity of • Halophiles: require high
surroundings salt concentrations
• Acidophiles grow optimally at pH below 5.5 • Marine bacteria ~3%
• Picrophilus oshimae (hot spring) has optimum pH of • Extreme halophiles ≥ 9%
less than 1! (thermoacidophilic) (Dead Sea, Utah’s salt flats)
• Alkaliphiles grow optimally at pH above 8.5
4.5. Nutritional Factors That Influence
Nutritional Factors Microbial Growth
§ Prokaryotes have remarkable metabolic diversity
Learning Outcomes § Require nutrients to synthesize cell components
• Lipid membranes, cell walls, proteins, nucleic acids
1. Distinguish chemically defined and complex • Made from subunits: phospholipids, sugars, amino acids,
media. nucleotides
2. Justify the use of each of the following: • Major elements, carbon, oxygen, hydrogen, nitrogen,
• sulfur, phosphorus, potassium, magnesium, calcium, and
anaerobic techniques, living host cells, candle iron
jars, selective and differential media,
enrichment medium.
4.5. Nutritional Factors That Influence 4.5. Nutritional Factors That Influence
§ Required elements § Growth factors
• Major elements make up cell components • Some microbes cannot synthesize certain molecules
• Carbon source distinguishes different groups • Amino acids, vitamins, purines, pyrimidines
• Heterotrophs use organic carbon • Only grow if these growth factors are available
• Autotrophs use inorganic carbon as CO2 (carbon fixation) • Reflects biosynthetic capabilities
• Nitrogen required for amino acids, nucleic acids • Escherichia. coli synthesizes all cellular components from
• Many use ammonia (some convert nitrate to ammonia) glucose, has wide metabolic capabilities
• Nitrogen fixation important • Neisseria unable to synthesize many, requires numerous
growth factors
• Iron, phosphorus often limiting
• Termed fastidious: have complicated nutritional
• Trace elements usually requirements, grow only if special nutrients are present.
available (cobalt, zinc, copper
molybdenum, manganese)
4.5. Nutritional Factors That Influence 4.5. Nutritional Factors That Influence
§ Energy sources § Nutritional diversity
• Sunlight, chemical compound • Photoautotrophs: energy from sunlight; carbon from CO2
• Photoheterotrophs: energy from sunlight; carbon from organic
§ Phototrophs obtain energy from sunlight compounds
• Plants, algae, photosynthetic bacteria • Chemolithoautotrophs (also termed chemoautotrophs,
chemolithotrophs): energy from inorganic compounds; carbon from
§ Chemotrophs extract energy from chemicals CO2
• Mammalian cells, fungi, many types of prokaryotes • Chemoorganoheterotrophs (also termed chemoheterotrophs,
chemoorganotrophs): energy and carbon from organic compounds
• Sugars, amino acids, fatty acids common sources
• Some prokaryotes use inorganic chemicals such as
hydrogen sulfide, hydrogen gas
4.6. Cultivating Microorganisms in the Laboratory 4.6. Cultivating Prokaryotes in the Laboratory
§ General categories of culture § Hundreds of types of

media media available
• Complex media contain a • Regardless, some
variety of ingredients medically important
• Exact composition highly microbes, and most
variable environmental ones,
• Often a digest of proteins have not yet been
• Chemically defined media grown in laboratory
composed of exact amounts of
pure chemicals
• Used for specific research
experiments
• Usually buffered
4.6. Cultivating Prokaryotes in the Laboratory 4.6. Providing Appropriate Atmospheric Conditions
§ Special types of culture media § Aerobic

• Useful for isolating and identifying • Most obligate aerobes and facultative anaerobes can be
a specific species incubated in air (~20% O2)
• Selective media inhibit growth of • Broth cultures shaken to provide maximum aeration
certain species • Many medically important bacteria (e.g., Neisseria,
• Differential media contain substance that microbes Haemophilus) grow best with increased CO2
change in identifiable way • Some are capnophiles, meaning require increased CO2
• One method is to incubate in candle jar
§ Microaerophilic
• Require lower O2 concentrations than achieved by
candle jar
• Can incubate in gas-tight container with chemical packet
• Chemical reaction reduces O2 to 5–15%
4.6. Providing Appropriate Atmospheric Conditions 4.6. Enrichment Cultures
§ Anaerobic: obligate anaerobes sensitive to O2 § Enrichment cultures used to isolate organism that
constitutes small fraction of mixed population
• Anaerobic containers useful if microbe can tolerate brief
O2 exposures; can also use semisolid culture medium • Provide conditions promoting growth of particular species
containing reducing agent (e.g., sodium thioglycolate) • E.g., specific carbon source
• Reduce O2 to water
• Anaerobic chamber provides more stringent approach
4.7. Methods to Detect and Measure
Methods to Detect and Measure Microbial Growth
Microbial Growth
Learning Objectives § Direct cell counts: total numbers (living plus dead)
• Direct microscope count
• Cell-counting instruments (Coulter counter, flow cytometer)
1. Explain the four direct methods of measuring cell
growth.
2. Differentiate direct and indirect methods of
measuring cell growth.
3. Explain three indirect methods of measuring cell
growth.
4.7. Methods to Detect and Measure 4.8. Methods to Detect and Measure
§ Viable cell counts: cells capable of multiplying • Plate counts determine colony-forming units (CFUs)
• Can use selective, differential media for particular species
• Plate counts: single cell gives rise to colony
• Plate out dilution series: 30–300 colonies ideal
4.8. Methods to Detect and Measure 4.8. Methods to Detect and Measure
• Membrane filtration • Most Probable Number (MPN)
• Concentrates microbes by filtration • Estimates cell concentration using dilution series
• Filter is incubated on appropriate agar medium • Sets of tubes are incubated; results are recorded and
compared to table to give statistical determination
Microbial Growth
§ Measuring biomass
• Turbidity is proportional to concentration of cells
• Measured with spectrophotometer

Microbial Growth
§ A wavelength of 600nm (A600) is used § Measuring biomass
for measuring bacterial concentration. The • Total weight can be measured
benefit of using a spectrophotometer is that it's • Tedious and time-consuming
quick and easy. The harvesting of a culture • Typically only used for filamentous organisms that do not
should be completed during the early log phase readily separate into individual cells for valid plate counts
• Cells in liquid culture centrifuged; pellet is weighed
of cell growth. When measuring the rate of
• Dry weight can be determined by heating pellet in oven
growth of bacteria in culture, an optical density
OD of .5-.7 indicates that the bacteria are in the § Detecting cell products
early to mid log phase of growth. It is best to • pH indicators
harvest the cells during this phase since the • Durham tubes (inverted tubes) to trap gas
bacteria are at their peak rate of growth. • CO2 production
• ATP production using enzyme luciferase to produce light
Reading Assignment: End
Direct and indirect counting/ measuring cell growth:
https://courses.lumenlearning.com/boundless-
microbiology/chapter/counting-bacteria/
THANK YOU…
Video on streak plate method:
https://www.youtube.com/watch?v=_1KP9zOtjXk
Chapter 05
MICROBIAL GROWTH Microbial Growth
CONTROL Control
Leila A. Ombat, PhD

Part 1 - Subtopics
1.A glimpse of history
2.Approaches to control
MICROBIAL GROWTH
3.Selection of antimicrobial procedure
CONTROL
(Part 1) 4.Using heat to destroy
microorganisms and viruses
Leila A. Ombat, PhD
5.Using other physical methods to
remove or destroy microbes
Part 2 - Subtopics A Glimpse of History
§ British Medical Journal stated British physician

5. Using chemicals to destroy microorganisms Joseph Lister (1827–1912)
and viruses “saved more lives by the introduction of his system
than all the wars of the 19th century together had
6. Classes of germicidal chemical sacrificed.”
7. Preservation of perishable products • Lister revolutionized surgery: introduced methods to
prevent infection of wounds
8. Contamination of an Operating Room • Impressed with Pasteur’s work, he wondered if
‘minute organisms’ might be responsible for infections
• Applied carbolic acid (phenol) directly onto damaged
tissues, where it prevented infections
• Improved methods further by sterilizing instruments and
maintaining clean operating environment
A Glimpse of History 5.1. Approaches to Control
§ Until late 19th century, patients undergoing even § Principles of Control (continued…)
minor surgeries were at great risk of developing • Decontamination: reduce pathogens to levels
fatal infections considered safe to handle (nearly 100% -
• Physicians did not know their hands could pass object/surface)
diseases from one patient to the next • Sanitized: substantially reduced microbial population
• Did not understand airborne microbes could infect that meets accepted health standards
open wounds • Not a specific level of control
§ Modern hospitals use strict • Preservation: process of delaying spoilage of foods

and other perishable products
procedures to avoid
• Adjust conditions
microbial contamination
• Add bacteriostatic (growth-inhibiting) preservatives
• Most surgeries performed
• Pasteurization: brief heating to reduce number of
with relative safety
spoilage organisms, destroy pathogens
• Food
5.1. Approaches to Control 5.1. Approaches to Control
§ Situational § Daily Life

considerations: • Washing and scrubbing with soaps and detergents
Microbial control achieves routine control
methods depend
• Soap aids in mechanical removal of organisms
upon situation and
• Beneficial skin microbiota reside deeper on underlying
level of control
layers of skin, hair follicles
required
– Not adversely affected by regular use
• Hand washing with soap and water most important step
in stopping spread of many infectious diseases
§ Hospitals § Microbiology Laboratories

• Minimizing microbial population very important • Routinely work with microbial cultures
• Danger of healthcare-associated infections (HAIs) • Use rigorous methods of control
• Patients more susceptible to infection • Must eliminate microbial contamination to both
• May undergo invasive procedures (surgery) experimental samples and environment
• Pathogens more likely found in hospital setting • Careful treatment both before (sterile media) and after
– Feces, urine, respiratory droplets, bodily secretions (sterilize cultures, waste)
• Instruments must be sterilized to avoid introducing • Aseptic techniques used to prevent contamination of
infection to deep tissues samples, self, laboratory
• Prions relatively new concern; difficult to destroy • Center for Disease Control guidelines for labs working
with microbes
• Biosafety levels range from BSL-1 (microbes not known
to cause disease) to BSL-4 (lethal pathogens for which
no vaccine or treatment exists)
§ Food and Food Production Facilities § Water Treatment Facilities

• Perishables retain quality longer when contaminating • Ensure drinking water free of pathogens
microbes destroyed, removed, inhibited • Chlorine traditionally used to disinfect water
• Heat treatment most common and reliable mechanism • Can react with naturally occurring chemicals
– Can alter flavor, appearance of products – Form disinfection by-products (DBPs)
• Irradiation approved to treat certain foods – Some DBPs linked to long-term health risks
• Chemical additives can prevent spoilage • Some organisms resistant to chemical disinfectants
• FDA regulates because of risk of toxicity – Cryptosporidium parvum (causes diarrhea)
• Facilities must keep surfaces clean and relatively free • Regulations require facilities to minimize DBPs and
of microbes C. parvum in treated water
§ Pharmaceuticals, cosmetics, deodorants must not
carry microbial contamination
5.2. Selection of an Antimicrobial Procedure
§ Selection of effective procedure is complicated

• Ideal method does not exist
• Each has drawbacks and procedural parameters
§ Choice depends on numerous factors
• Type and number of microbes
• Environmental conditions
• Risk of infection
• Composition of infected item
5.2. Selection of an Antimicrobial Procedure 5.2. Selection of an Antimicrobial Procedure

Protozoan cysts and oocysts: resistant to disinfectants;
§ Type of Microorganism excreted in feces; causes diarrheal disease if ingested
• Multiple highly resistant microbes
• Bacterial endospores: most resistant, only extreme
heat or chemical treatment destroys them resistant to
disinfectants
Mycobacterium species: waxy cell walls makes resistant to many Pseudomonas species: resistant to and can actually grow in
chemical treatments some disinfectants
Non-enveloped viruses: lack lipid envelope; more resistant § Number of Microorganisms

to heat. • Time for heat, chemicals to kill affected by population
size
• Fraction of population dies during given time interval
• Large population = more time
• Removing organisms by
washing reduces time
• Decimal reduction time
(D value) gauges
commercial effectiveness
• Time required to kill 90% of
population under specific
conditions
§ Environmental Conditions § Risk for Infection

• Medical instruments categorized according to
• Dirt, grease, body fluids can interfere
risk for transmitting infectious agents
with heat penetration, action of
• Critical items come in contact with body tissues
chemicals
• Must be sterile
• Important to thoroughly clean
• Include needles and scalpels
• Microorganisms in biofilm are more • Semicritical instruments contact mucous
resistant membranes but do not penetrate body tissues
• pH, temperature can influence • Must be free of viruses and vegetative bacteria
effectiveness • Few endospores blocked by mucous membranes
• For example, sodium hypochlorite • Includes endoscopes and endotracheal tubes
(household bleach) solution can kill • Non-critical instruments contact unbroken skin
suspension of M. tuberculosis at only
55°C in half the time as at 50°C • Low risk of transmission
• Even more effective at low pH • Countertops, stethoscopes, blood pressure cuffs
5.3. Using Heat to Destroy Microorganisms
5.2. Selection of an Antimicrobial Procedure and Viruses
§ Composition of Item § Heat treatment useful for microbial control
• Some sterilization and disinfection methods • Reliable, safe, relatively fast, inexpensive, non-toxic
inappropriate for certain items • Can be used to sterilize or disinfect
• Heat inappropriate for plastics and other sensitive items
§ Moist heat: irreversibly denatures proteins
• Irradiation provides alternative, but damages some types
of plastic • Boiling destroys most microorganisms and viruses
• Moist heat, liquid chemical disinfectants cannot be used • Does not sterilize: endospores can survive
to treat moisture-sensitive material • Pasteurization destroys heat-sensitive pathogens,
spoilage organisms
• High-temperature–short-time (HTST): most products
– Milk: 72°C for 15 s; ice cream: 82°C for 20 s
• Ultra-high-temperature (UHT): shelf-stable boxed juice
and milk; known as “ultra-pasteurization”
– Milk: 140°C for a few seconds, then rapidly cooled
5.3. Using Heat to Destroy Microorganisms 5.3. Using Heat to Destroy Microorganisms
and Viruses and Viruses
§ Sterilization Using Pressurized Steam § Commercial Canning Process
• Autoclave used to sterilize using pressurized steam • Uses industrial-sized autoclave called retort
• Increased pressure raises temperature; kills endospores • Designed to destroy Clostridium botulinum endospores
• Sterilization typically at 121°C and 15 psi in 15 minutes • Reduce 1012 endospores to only 1 (a 12 D process)
– Longer for larger volumes • Virtually impossible to have so many endospores
• Flash sterilization at higher
• Critical because otherwise endospores can germinate
temperature can be used
in canned foods; cells grow in low-acid anaerobic
• Prions thought destroyed conditions and produce botulinum toxin
at 132°C for 1 hour
• Canned food commercially sterile
• Endospores of some thermophiles may survive
• Usually not a concern; only grow at temperatures well
above normal storage
5.3. Using Heat to Destroy Microorganisms

Retort sterilization and Viruses
§ Dry heat
• Less effective than moist heat; longer times, higher
temperatures necessary
• 200°C for 90 minutes vs. 121°C for 15 minutes
• Hot air ovens oxidize cell components, denature proteins
• Used for glass, powders, oils, dry materials
• Incineration a method of dry heat sterilization
• Oxidizes cell to ashes
• Used to destroy medical waste and animal carcasses
• Laboratory inoculation loop sterilized by flaming
5.4. Using Other Physical Methods to Remove
or Destroy Microbes
§ Some materials cannot withstand heat treatment
§ Filtration retains bacteria
• Filtration of fluids used extensively
• Membrane filters
– Small pore size (0.2 µm) to remove bacteria
– Thin
• Depth filters
– Thick porous filtration
material (e.g., cellulose)
– Larger pores
– Electrical charges trap cells
• Filtration of air
• High-efficiency particulate air (HEPA) filters remove
nearly all microbes from air
5.4. Using Other Physical Methods to Remove

Biosafety cabinet, Class II
or Destroy Microbes
§ Radiation
• Electromagnetic radiation: radio waves, microwaves,
visible and ultraviolet light, X rays, and gamma rays
• Energy travels in waves; no mass
• Wavelength inversely proportional to frequency
• High frequency has more energy than low frequency
5.4. Using Other Physical Methods to Remove 5.4. Using Other Physical Methods to Remove
or Destroy Microbes or Destroy Microbes
§ Radiation (continued…) § Radiation (continued…)
• Ionizing radiation can remove electrons from atoms • Ultraviolet radiation destroys microbes directly
• Gamma rays and X rays important forms • Damages DNA
• Destroys DNA • Used to destroy microbes in air, water, and on surfaces
• Damages cytoplasmic membranes • Poor penetrating power
• Reacts with O2 to produce reactive oxygen species – Thin films or coverings can limit effect
• High energy gamma-rays – Cannot kill microbes in solids or turbid liquids
– Used to sterilize heat-sensitive materials – Most glass and plastic block
– Generally used after packing • Must be carefully used since damaging to skin, eyes
– Approved for use on foods, although consumer resistance
• Microwaves kill by generated heat, not directly
has limited use
– FDA has approved for spices and dried herbs, fruits, • Microwave ovens heat food unevenly, so cells can survive
vegetables, and grains (insect control), pork (parasite
control), and poultry, beef, lamb, and pork (bacterial control)
5.4. Using Other Physical Methods to Remove 5.4. Using Other Physical Methods to Remove
or Destroy Microbes or Destroy Microbes
§ High Pressure
• Used in pasteurization of commercial foods
• For example, guacamole (Avocado-based dip)
• Avoids problems with high temperature pasteurization
• Employs high pressure up to 130,000 psi
• Destroys microbes by denaturing proteins and altering cell
permeability
• Products maintain color, flavor associated with fresh food
Part 2 - Subtopics
5. Using chemicals to destroy microorganisms

MICROBIAL GROWTH and viruses
CONTROL 6. Classes of germicidal chemical
(Part 2) 7. Preservation of perishable products
8. Contamination of an Operating Room
Leila A. Ombat, PhD
5.5. Using Chemicals to Destroy Microorganisms 5.5. Using Chemicals to Destroy Microorganisms
and Viruses and Viruses
§ Potency of Germicidal Chemical Formulations § Selecting the Appropriate Germicidal Chemical
• Sterilants destroy all microorganisms also called sporocides • Toxicity: benefits must be weighed against risk of use
• Heat-sensitive critical instruments (cut bone and/or penetrate
soft tissue, carry the highest risk of disease transmission) • Activity in presence of organic material
• High-level disinfectants destroy viruses, vegetative cells • Many germicides inactivated
• Do not reliably kill endospores • Compatibility with material being treated
• Semi-critical instruments (touch mucous membrane /non-intact • Liquids cannot be used on electrical equipment
skin, lower risk of disease transmission)
• Residues: can be toxic or corrosive
• Intermediate-level disinfectants destroy vegetative bacteria,
mycobacteria, fungi, and most viruses • Cost and availability
• Disinfect non-critical instruments (contact intact skin, lowest risk) • Storage and stability
• Low-level disinfectants destroy fungi, vegetative bacteria except • Concentrated stock decreases storage space
mycobacteria, and enveloped viruses
• Environmental risk
• Do not kill endospores, non-enveloped viruses
• Disinfect furniture, floors, walls
• Agent may need to be neutralized before disposal
5.5. Classes of Germicidal Chemicals 5.5. Classes of Germicidal Chemicals
§ Alcohols § Aldehydes
• 60–80% aqueous solutions of ethyl or isopropyl alcohol • Glutaraldehyde, formaldehyde, and ortho-
• Kills vegetative bacteria and fungi phthalaldehyde (OPA)
• Not reliable against endospores, some naked viruses • Inactivates proteins and nucleic acids
• Coagulates essential proteins (e.g., enzymes) • 2% alkaline glutaraldehyde common sterilant
• More soluble in water; pure alcohol less effective • Immersion for 10–12 hours kills all microbial life
• Damage to lipid membranes • Toxic
• Commonly used as antiseptic and disinfectant • Formaldehyde used as gas or as formalin (37% solution)
• Limitations • Effective germicide that kills most microbes quickly
• Evaporates quickly, limiting contact time • Used to kill bacteria and inactivate viruses for vaccines
• Can damage rubber, some plastics, and others • Used to preserve specimens
§ Biguanides § Ethylene oxide

• Chlorhexidine most effective • Useful gaseous sterilant
• Extensive use as antiseptics • Destroys microbes including endospores and viruses
• Stays on skin, mucous membranes • Reacts with proteins
• Relatively low toxicity • Penetrates fabrics, equipment, implantable devices
• Destroys vegetative bacteria, fungi, some enveloped viruses • Pacemakers, artificial hips
• Common in many products: skin cream, mouthwash
• Useful in sterilizing heat- or moisture-sensitive items
• Many disposable laboratory items
• Petri dishes, pipettes
• Applied in special chamber resembling autoclave
• Limitations: toxic (must be extensively aired), mutagenic
and potentially carcinogenic
§ Halogens: oxidize proteins, cellular § Metal Compounds

components • Combine with sulfhydryl groups of proteins
§ Chlorine: Destroys all microorganisms and viruses
• High concentrations too toxic to be used medically
• Used as disinfectant
• Caustic to skin and mucous membranes • Silver still used as an antiseptic: creams, bandages
• 1:100 dilution of household bleach effective • Silver nitrate eye drops given at birth to prevent Neisseria
• Very low levels disinfect drinking water gonorrhoeae infections
• Cryptosporidium oocysts, Giardia cysts survive • Replaced by antibiotics
• Organic compounds interfere • Compounds of mercury, tin, copper, and others once
• Chlorine dioxide used as disinfectant and sterilant widely used as preservatives
§ Iodine: Kills vegetative cells, unreliable on endospores • In industrial products
• Used as iodophore
• To prevent microbial growth in recirculating cooling water
• Iodine slowly released from carrier molecule
• Extensive use led to environmental pollution
• Pseudomonas species can survive in stock
solution • Now strictly regulated
§ Ozone (O3) § Peroxygens: powerful oxidizers used as sterilants

§ Unstable form of oxygen • Readily biodegradable, no residue
• Decomposes quickly, so generated on-site • Less toxic than ethylene oxide, glutaraldehyde
• Powerful oxidizing agent • Hydrogen peroxide: effectiveness depends on surface
• Used as alternative to chlorine • Aerobic cells produce enzyme catalase
• Disinfectant for drinking and wastewater – Breaks down H2O2 to O2, H2O
• More effective on inanimate object
• Doesn’t damage most materials
• Hot solutions used in food industry
• Vapor-phase can be used as sterilant
• Peracetic acid: more potent than H2O2
• Effective on organic material
• Useful on wide range of material
§ Phenolic Compounds (Phenolics) § Quaternary Ammonium Compounds (Quats)

• Cationic detergents
• Phenol one of earliest disinfectants
• Low toxicity, disinfection of food preparation surfaces
• Has unpleasant odor, irritates skin • Charged hydrophilic and uncharged hydrophobic regions
• Phenolics kill most vegetative bacteria • Reduces surface tension of liquids
• Mycobacterium at high concentrations • Aids in removal of dirt, organic matter, organisms
• Not reliable on all virus groups • Most household soaps, detergents are anionic
• Destroy cytoplasmic membranes, denature proteins • Positive charges of quats attracted to negative charges of cell
surface
• Wide activity range, reasonable cost, remain effective in • Reacts with membrane
presence of detergents and organic contaminants • Destroys vegetative bacteria and enveloped viruses
• Leave antimicrobial residue • Not effective on endospores, mycobacteria, non-enveloped
• Considered non-toxic for skin applications, use viruses
cautioned by FDA and EPA (Evi. Protection Agency) • Pseudomonas resists, can grow in solutions
• Triclosan, hexachlorophene
5.5. Classes of Germicidal Chemicals 5.6. Preservation of Perishable Products
§ Chemical preservatives
• Food preservatives must be non-toxic for safe ingestion
§ Weak organic acids (benzoic, sorbic, propionic)
• Alter cell membrane function
• Control molds and bacteria in foods
§ Nitrate and nitrite used in processed meats
• Inhibit endospore germination and vegetative cell growth
• Stops growth of Clostridium botulinum
• Higher concentrations give meats pink color
• Shown to be carcinogenic—form nitrosamines
5.6. Preservation of Perishable Products 5.6. Preservation of Perishable Products
§ Low-Temperature Storage § Reducing Available Water

• Refrigeration inhibits growth of pathogens and spoilage • Accomplished by salting, adding sugar, or drying food
organisms by slowing or stopping enzyme reactions • Addition of salt, sugar increases environmental solutes
• Psychrotrophs, psychrophilic organisms can still grow • Causes cellular plasmolysis (water exits bacterial cells)
• Freezing preserves by stopping all microbial growth • Some bacteria grow in high salt environments
• Some microbial cells killed by ice crystal formation, but • Staphylococcus aureus
many survive and can grow once thawed
• Drying often supplemented by salting
• Lyophilization (freeze drying) foods
• Coffee, milk, meats, fruits, vegetables
• Drying stops microbial growth but does
not reliably kill
• Numerous cases of salmonellosis from
dried eggs
Contamination of an Operating Room

END
§ Contamination occurs readily
§ Cleaning afterwards critical
THANK YOU…

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Microbial Nutrition & Microbial Nutrition

Microbial Growth MICROORGANISMS REQUIRE A SUITE OF

PREPARED BY: FLORENCE JHUN F. ALMADIN

Microbial Nutrition Microbial Nutrition

Microbial Nutrition Microbial Nutrition

Macronutrients = needed in relatively larger quantities

Examples: C, O, N, H (predominantly required)

PREPARED BY: FLORENCE JHUN F. ALMADIN

Microbial Nutrition Microbial Nutrition

Microbial Nutrition Microbial Nutrition

A. Synthetic culture media: B. Complex culture media:

Microbial Nutrition Microbial Nutrition

Microbial Nutrition MSA: Selective and Differential

PREPARED BY: FLORENCE JHUN F. ALMADIN

Microbial Nutrition Blood Agar

Triple Sugar Iron Test Antibiotic Sensitivity Testing

Microbial Nutrition Microbial Nutrition

20 agar stabs x 10 ml/stabs = 200 ml For Suc 7.0 g = xg

Total Volume to be prepared = 660 ml For Agar 1.5 g = xg = 9.90 g

For dH2O 660 ml

Preparation of Culture Media

For Agar 1.5 g = Xg = 6.40 g

Preparation of Culture Media

Preparation of Culture Media

- Orderly increase of all the chemical constituents of an organism

Ø For most microorganisms: growth followed by cell division

Microbial Growth Microbial Growth

ØOrganisms that divide by • Lag Phase

Microbial Growth Microbial Growth

1. Lag phase 3. Stationary phase

Typical Bacterial Growth Curve Typical Bacterial Growth Curve

Environmental Factors That Influence

• Prokaryotes inhabit nearly all environments Environmental

4.4. Temperature Requirements Temperature Requirements

Measuring Microbial Growth Measuring Microbial Growth

Ways to measure microbial growth:

Measuring Microbial Growth Measuring Microbial Growth

Measuring Microbial Growth Measuring Microbial Growth

Methods employed in inoculating

CFU/ml = 234+ 34+ 25 5 CFU/ml = 234+ 34+ 25 5 CFU/ml = 2.4 X 107

CFU/ml = 104.5 x 1x104 CFU/ml = 104.5 x 1x104 CFU/ml = 1.04 X 107

Measuring Microbial Growth Measuring Microbial Growth

Measuring Microbial Growth Measuring Microbial Growth

Measuring Microbial Growth Standard Curve

Standard Curve Standard Curve

Get the cell population

Control Tubes OD Reading Cells/ml at a OD Reading

Plot in Excel (Linear Equation)

Standard Curve Standard Curve

Can vary depending on the

Growth Curve Growth Curve

v Increase in cell number that occurs in an exponentially

Quantitative aspects of Microbial Growth Quantitative aspects of Microbial Growth

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§ Prokaryotes can be found growing in severe

4.1. Principles of Bacterial Growth 4.1. Principles of Bacterial Growth

§ Prokaryotic cells divide by § Growth can be calculated

4.1. Principles of Bacterial Growth 4.2. Prokaryotic Growth in Nature

§ The power of exponential growth § Microorganisms historically studied in laboratory