Basic Microbiology

David Matsuhiro
Cleanroom Compliance Inc.
dmatsuhiro@cleanroomcompliance.com

Microbiology 101
 Microbiology: The study of microorganisms
that exist as single cells or cell clusters. A
single microbial cell is generally able to
carry out the life process of growth,
respiration and reproduction, independent
of other cells of the same or of different
kinds.

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 Microbiology 101
 Concentrations of micro-organisms
 Soil – Cubic centimeter (1/5 teaspoon)

can contain as much as100-million CFU

 One acre of soil to a depth of 1 meter

can contain 3000 pounds of bacteria

 One square centimeter of the tung

contain 100-million CFU

 Fecal matter can contain 1-billion CFU

Microbiology 101
 Concentrations of micro-organisms
 Normal day a person can shed 2-3

million bacteria
 A sneeze can spray as much as 250-

thousand CFU

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 Microbiology 101
 Prokaryotic Cells: Do not have a true
nucleus, however their heredity material is
contained within a single DNA molecule
 Bacteria: Simplest living organisms with

over 2,000 species

 Most common contaminant of classified

pharmaceutical environments
 Blue-green algae: Contain two forms of

chlorophyll and are primary aquatic

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Microbiology 101
 Eukaryotic Cells: Have true nucleus within
the cell
 Algae: A group of chlorophyllus

microorganisms
 Protozoa: Unicellular organism that lacks

a true cell wall and obtains its nutrition

phagotrophically

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 Microbiology 101
 Eukaryotic Cells Continued
 Fungi: microorganisms that have rigid cell

walls, lack chlorophyll and grows on

organic matter parasitically. Second most
common contaminant of classified
pharmaceutical environments
 Viruses: Require a host cell for replication

Microbiology 101
 Growth Requirements
 Temperature is one of the most important
environmental factors influencing the
growth and survival of organisms
 Minimum Temperature: Temperature at

which growth no longer occurs

 Optimum Temperature: Temperature at

which growth is most rapid

 Maximum Temperature: Temperature
above which growth is not possible
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 Microbiology 101
 Temperature
 Stenothermal: Organisms with narrow

temperature growth range

 Psychrophiles: Low temperatures for
optimal growth, 0 to 20 °C
 Mesophiles: Mid temperatures for
optimal growth, 15 to 45 °C
 Thermopiles: High temperatures for
optimal growth, 40 to 70 °C
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Microbiology 101
 Temperature
 Extreme Conditions

 Thermal ocean vents (765ºF/407ºC)

 Sahara desert (150ºF/66ºC)

 Antarctica (-132ºF/-91ºC)

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 Microbiology 101
 Temperature

02 - 08°C 20 - 25°C
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Microbiology 101
 Temperature

30 - 35°C
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 Microbiology 101
 Oxygen Requirements
 Obligate Aerobes: Requires oxygen
because they are unable to generate
energy by fermentation
 Facultative Organisms: Able to obtain
energy by either respiration or
fermentation
 Microaerophilic: Requires minimal
oxygen levels

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Microbiology 101
 Oxygen Requirements
 Aero-tolerant Anaerobes: Don’t use
oxygen but are not harmed by it
 Obligate Anaerobes: Do not use
oxygen and are harmed by it
 Extreme Conditions

 High elevations of mount Everest

 Ocean depths of the Atlantic Ocean

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 Microbiology 101
 Oxygen Requirements

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Microbiology 101
 Nutrient concentrations can affect both
growth rate and total growth
 Low Nutrient: At very low nutrients, the

microbial growth rate is reduced

 Moderate Nutrients: Optimal growth
rate with maximum total growth
 High Nutrients: May have reduced
growth rate and the total growth may be
diminished
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 Microbiology 101
 Nutrient concentrations
 Extreme Conditions

 High quality water such as WFI

 High sugar content foods and

products

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Microbiology 101
 Nutrients

R2A - Low MPCH - Low

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 Microbiology 101
 Nutrients

Cornmeal - Medium TSA - High

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Microbiology 101
 Nutrients

Rose Bengal Baird Parker

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 Microbiology 101
 Nutrients

Blood Agar SDA

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Microbiology 101
 Nutrients

Manitol Manitol Salt

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 Microbiology 101
 Chemical Conditions
 The term pH refers to the acidity or

alkalinity of a liquid
 The pH scale ranges from 0 – 14

 Most bacteria have optimum growth at

a pH of 6.5 – 7.5
 Ranges of growth occur from a pH of

2.0 - 11

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Microbiology 101
 Approximate pH of common liquids
 Lime juice, 2

 Vinegar, 3

 Saliva, 6

 Water, 7

 Egg, 8

 Draino, 14

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 Microbiology 101
 Humidity
 Most microbes like a humidity of about

65%
 The lower the humidity, the faster the

plates dry out. This has a significant

impact on incubation time

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Microbiology 101
 Bacterial Growth
 Bacteria increase in numbers by binary

fission
 One cell divides into two

 Two divides into four

 Four divided into eight

 Final population is equal to 2

N

N is the number of times the

population doubles
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 Microbiology 101
 Bacterial Growth
 Generation time is the time it takes an

organism to double in minutes

 Bacillus stearothermophilus, 11

 Escherichia coli, 20

 Lactobacillus acidophilus, 75

 Mycobacterium tuberculosis, 360

 Treponema pallidum, 2000

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Microbiology 101
 Bacterial Growth
 Generation time is the time it takes an

organism to double in minutes

 Bacillus stearothermophilus, 11
 Escherichia coli, 20
 Lactobacillus acidophilus, 75
 Mycobacterium tuberculosis, 360
 Treponema pallidum, 2000

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 Microbiology 101
 Growth Rate
 The growth curve for all organisms is

relatively the same. They are

composed of the following phases.
 Lag Phase:

 The period after inoculation

before significant growth begins
 Acclimating to environment

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Microbiology 101
 Log Phase:
 Period in which growth increases

at an exponential rate
 Generation time is based on
growth conditions

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 Microbiology 101
 Stationary Phase
 Curve rapidly flattens out of the

growth curve
 The depletion of growth factors

and accumulation of toxic waste

 Period in which the cycle of
growth ceases or the number of
cells dividing equals to the
number that are dying
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Microbiology 101
 Death Phase
 Period in which the number of

dying cells is more that 50%

 The viable cells begins to drop

exponentially
 Complete death phase is based

on the individual organism and

type of nutrient source

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 Microbiology 101
Microbial Growth Curve

CFU’s Stationary Phase

Log Phase Death Phase

Time

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Microbiology 101
 Microbial Isolation

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 Microbiology 101
 Macroscopic Evaluation
 Form/Shape

 Elevation

 Margin

 Color

 Size Consistency

 Surface Characteristics

 Optical

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Microbiology 101
 Macroscopic Evaluation

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 Microbiology 101
 Macroscopic Evaluation

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Microbiology 101
 Macroscopic Evaluation

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 Microbiology 101
 Macroscopic Evaluation

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Microbiology 101
 Microscope
 Human eye capability 100 µm

 Simple microscope is composed of

only one lens
 Compound microscope uses two or

more lenses
 Practical magnification limit is
approximately 1,000 – 2,000

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 Microbiology 101
 Microscope Limiting Characteristics
 Resolution is the ability to separate

closely spaced objects

 Wavelength of light is another factor

 Light microscopes use visible light with

the following wave lengths

 Deep violet (400 nm)

 Deep red (700 nm)

 Bacteria measures 1 um (100 nm)

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Microbiology 101
 Microscope Limiting Characteristics
 Electron Microscope

 Illuminating objects with a beam of

electrons
 This beam has significantly shorter

wave lengths
 Sample must be viewed under high

vacuum which may cause distortion

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 Microbiology 101
 Microscope Limiting Characteristics
 Scanning Electron Microscopy

 Provides a three dimensional picture

of the cell
 Tunneling Electron Microscopy

 Scans the surface and a probe

converts the movement to a monitor

screen
 Most recent advancement with
extremely high resolution 43

Microbiology 101
 Bacterial Characteristics
 Composed of approximately 70% water

 Appear translucent in its natural state

 Stains can be used to optimize viewing of

the cells under magnification

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 Microbiology 101
 Bacterial Cell Characteristics

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Microbiology 101
 Gram Stain
 Differential staining method

 benchmark used in the ID of bacteria

 Reactions of cells to stain based on

fundamental differences in physical

structures
 Both have rigid cell wall and outer
membranes
 Membrane controls what enters and
leaves cell 46

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 Microbiology 101
 Gram Stain
 Differential staining method

 Most commonly used stain in micro

 Used to evaluate microscopic features

 Can be difficult to evaluate

 Optimum one cell layer thick

 Gram Positive organisms, blue to purple

 Gram Negative organisms, red to pink

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Microbiology 101
 Sizes of bacteria
 Unit Length

 Micron, µ, 1-millionth of a meter

 1/25,400 of an inch

 Micrometer, µm

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 Microbiology 101
 Common Particulate Sizes

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Microbiology 101
Gram Stain

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 Microbiology 101
 Microscopic shapes

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Microbiology 101
 Basic Shapes
 Cocci

 Spheres or balls

 Bacilli

 Rods or tubular

 Spirilla

 Spirals or cork screw

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 Microbiology 101
 Gram Positive Organisms
 Cocci

 Grape like clusters

 Pearl strands

 Tetrads

 Rods

 Small

 Large

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Microbiology 101
Gram Positive Cocci Macroscopic

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 Microbiology 101
Gram Positive Cocci grape like clusters
Staphylococcus

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Microbiology 101
Gram Positive Cocci tetrad clusters
Micrococcus

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 Microbiology 101
Gram Positive Cocci pearl strands
Streptococcus

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Microbiology 101
Gram Positive Small Rod Macroscopic

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 Microbiology 101
Gram Positive Small Rod
Corynebacterium

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Microbiology 101
Gram Positive Bacillus Macroscopic

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Microbiology 101
Gram Positive Rod Large
Bacillus

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Microbiology 101
Gram Positive Rod Large
Bacillus

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Microbiology 101
Gram Positive Rod Large
Bacillus

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Microbiology 101
Bacillus

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 Microbiology 101
Gram Negative Rods Macroscopic

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Microbiology 101
Gram Negative Organisms
 Rods

 Small

 Coccobacillus (Niseria)

 Spiral or Corkscrew (Campylobacter)

 Curved or crescent (Vribrio)

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Microbiology 101
Gram Negative Rods
E. coli

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Microbiology 101
Gram Negative Rods
E. coli

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 Microbiology 101
Too numerous To Count
(TNTC)

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Microbiology 101
Unable To Enumerate
(UTE)

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 Microbiology 101
 Micro plates are providing useful
information, but most people do not know
or understand how to interpret the results
 The results are not just a number

 It’s important to understand the types or

organisms
 Assess the type of sampling methods

and techniques

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Microbiology 101
 The following plates were taken from my
fingers after working in the lab. Please
match the following samples with the
pictures
 Dirty hands

 Water rinse

 Soap

 Bactericidal soap

 Alcohol foam

 Alcohol gel

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 Microbiology 101
Finger Samples

______________ _______________
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Microbiology 101
Finger Samples

______________ _______________
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 Microbiology 101
Finger Samples

______________ _______________
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Microbiology 101
 Takeaway Message
 Gram Positive: People

 Gram Negative: Water

 Bacillus: Forms spores and difficult to kill

 Micro-organisms are everywhere!

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