Microbiology Test 1- Mic 101

Front 1

Gram Positive Bacteria
Micrococcus

Back 1

ococcus
• Spherical Bacteria (Cocci [G= berry])
• Obligate Aerobe
• Common in Soils and on Human Skin
• Almost always brightly Pigmented
- Micrococcus luteus.
Micrococcus luteus (L= yellow) is a very common Contaminant in Lab. The Pigment acts as Sunscreen to protect the Bacteriumʼs DNA.

Front 2

Gram Positive Bacteria
Staphylococcus

Back 2

• Spherical Bacteria that grow in Clusters
• Facultative Aerobe
• Simple Nutritional Requirements (compared with Streptococci). - Staphylococcus epidermidis
All Humans have Staphylococcus epidermidis as their Part of their normal Skin Flora. It is almost always Harmless. Roughly 25% of Humans also have Staphylococcus aureus as part of their normal Skin Flora. And 40% of Humans who have Staphylococcus aureus
have Methicillin-Resistant Staphylococcus aureus (MRSA). It is particularly common Cause of Nosocomial Infections (G= person attending sick).

Front 3

Gram Positive Bacteria
Streptococcus

Back 3

• Spherical Bacteria that grow in Strips
• Aerotolerant Anaerobes
• Very Complex Nutritional Requirements
- Streptococcus lactis
Streptococcus lactis is used in the Production of Natural Cheeses.

Front 4

Gram Positive Bacteria
Endospore Formers
• Aerobic Endospore Formers

Back 4

- Bacillus cereus
Bacillus is a very common Bacterium in Soil and youʼll probably end-up isolating several Dozen different Bacillus Species in your Soil Sample. Saying “Bacillus” is like saying “Canis” in that itʼs one of the largest and
most diverse of Genera and as such includes the Bacterial Equivalent of a Range from Chihuahuas to Dachshunds to German Shepherds.

Front 5

Gram Negative Bacteria
Pseudomonads

Back 5

• Obligate Aerobic Rods
- Nonfermenters -- Utilize Oxygen as the Terminal Electron Acceptor
• Most are Motile via Polar Flagella
- Pseudomonas putida
Pseudomonas is commonly found in Soils. Pseudomonads can utilize an unusually wide Variety of Organic Compounds as Carbon and Energy Sources. They produce characteristic fluorescent Siderophores (G= star [iron] holders) to sequester (L= commit for safekeeping) Iron, commonly a Rate-Limiting Nutrient in Soils.

Front 6

Gram Negative Bacteria
Enterics

Back 6

• Small, Non-Spore-Forming Rods
• Facultative Aerobes
• Simple Nutritional Requirements
• Usually Motile via Peritrichous Flagella
- Ferment Glucose under Anaerobic Conditions
Since the Identification of Enterics can be Medically Important, there are numerous Diagnostic Tests for distinguishing the different Species of Enterics.

Front 7

Gram Negative Bacteria
Enterics
Mixed-Acid Fermenter

Back 7

• Mixed-Acid Fermentation of Glucose produces large Amounts of Acids
- Escherichia coli (E. coli) [Travelerʼs Diarrhea]
E. coli is the best-known Bacterium on this Planet. It grows in the Gut --usually as a Non-Pathogen -- and since it cannot normally survive for long outside of the Gut it serves as an Important Indicator Bacterium for Fecal Contamination.

Front 8

Gram Negative Bacteria
Enterics
Butanediol Fermenter

Back 8

• Butanediol Fermentation of Glucose produces Non-Acidic Compounds
- Enterobacter aerogenes
Enterobacter is routinely found in the Soil but can also take-up Residence inside the Gut. Water Testing tends to be obsessed with distinguishing Enterobacter (a Native Inhabitant of the Soil) from E. coli (an Indicator Bacterium for Fecal Contamination).

Front 9

• Bacteria found in Soil

Back 9

- Pseudomonas putida
Gram Negative Obligate Aerobic Rods
- Bacillus cereus
Gram Positive Facultative Aerobic Endospore-Forming Rods

Front 10

• Bacteria found in Food

Back 10

- Streptococcus lactis
Gram Positive Aerotolerant Anaerobic Spheres

Front 11

• Human Flora (Bacteria found on or in Humans)
Skin Bacteria

Back 11

- Micrococcus luteus
Gram Positive Obligate Aerobic Spheres
- Staphylococcus epidermidis
Gram Positive Facultative Aerobic Spheres

Front 12

• Human Flora (Bacteria found on or in Humans)
Enteric Bacteria (Bacteria found in the Enteric Tract)

Back 12

- Enterobacter aerogenes
Gram Negative Facultative Aerobic Rods
- Escherichia coli (E. coli)
Gram Negative Facultative Aerobic Rods

Front 13

Bacteria (G= rod)
• Bacteria are small (very small)

Back 13

A Typical Bacterium (E. coli) measures approximately 1 X 2 µm.
A Typical Eukaryotic Cell measures approximately 30 µm in Diameter.

Front 14

• Bacteria are Prokaryotes (G= before a kernel [nucleus]) whose Shapes are
determined by a uniquely structured Cell Wall

Back 14

- Prokaryotes do not have a Membrane-bounded Nucleus or
Membrane-bounded Organelles

Front 15

• Metabolic Functions in Bacteria occur within different Areas within the
same Compartment

Back 15

- The Cell Membrane contains Enzymes for generating ATP from
Aerobic Respiration
• Bacteria set-up Proton Gradients for ATP Synthesis
by translocating H+ out of the Cell. This is very similar to how Eukaryotic Mitochondria set-up Proton Gradients for ATP Synthesis by translocating H+ out of Mitochondria.

Front 16

• The Nucleoid (G= like a nucleus) is specialized for Information Storage,
Processing and Distribution

Back 16

- A Typical Bacterial Genome is 6 to 10 Mbp
The Nucleoid is essentially analogous to a Eukaryotic Nucleus.

Front 17

The Germ Theory (1863)

Back 17

• Pasteur proposed his Germ Theory
(1) Specific Microbes have specific Metabolic Processes
(2) Microbes are present in the Air
(3) Specific Microbes can cause specific Diseases in Wine

Front 18

• Koch published his Opus on Tuberculosis in 1884 proving Pasteurs Germ Theory

Back 18

• Kochʼs Postulates:
1- The Microbe must be found in a diseased Animal but not in a healthy Animal
2- The Microbe must be isolated and grown in Pure Culture
3- The Microbe from this Pure Culture must produce the same Disease when inoculated into healthy Animals
4- The Microbe must be re-isolated from these newly diseased Animals

Front 19

Population Growth: The Bacterial Growth Curve

Back 19

• An increase in Cellular Constituents Itʼs inconvenient to follow individual Microbes
- Itʼs more convenient to follow Populations

Front 20

Bacteria are usually grown in a Closed System
Batch Culture

Back 20

• No Fresh Medium is added
- Nutrients will be depleted
- Waste Products will accumulate

Front 21

- Lag Phase

Back 21

• Period during which Bacteria adapt to their new Environment
• No immediate increase in Cell Mass or Cell Numbers
• Highly Variable Time-wise
- Can be short to non-existent in Young Cultures in the same Medium
- Can be quite long when the Starter Culture is an old Culture, a Refrigerated Culture, or a Culture shifted into a different Medium
• New Cell Component and ATP Synthesis
• Microbes may need to “re-tool” for new Medium
In vivo, this is the Ideal Time for Host Defenses to kick-in. The Bacterial Population is at its most Vulnerable.

Front 22

- Logarithmic Phase (Exponential Phase)

Back 22

• Period during which Bacteria undergo rapid Binary Fission
• Maximum Growth possible for a particular Bacterium under a given set of Conditions (usually -- but not always -- 37°C)
- Constant Rate of Growth
This results in a J-Shaped Curve if the Actual Numbers are Graphed or a Straight Line if the Logarithms of the Numbers are Graphed .
- Culture is at its most Uniform
• Best time for Biochemical Studies (i.e. Identification)
In vivo this is when Disease Symptoms first Appear. The Bacteria are dividing too rapidly for easy Elimination. The Bacteria are sufficiently Numerous to alter their Environment and induce Damage in the Host.

Front 23

Stationary Phase (Spell it with an “E” if it goes into an Envelope)

Back 23

• Period during which Death Rate equals Growth Rate
• Population Growth ceases
- The Number of Cell Divisions equals the Number of Cell Deaths -- or --- No Cell Divisions
• Nutrient/O2 Depletion and Accumulation of Waste Products In vivo this is when Host Defenses can once again become Effective. This is why even if you do nothing, the Patient will usually get better. This is the Rationale behind the Maxim “First Do No Harm.

Front 24

- Death Phase

Back 24

• Period during which Death Rate exceeds Growth Rate
• Growth Curve goes Downward
- The Number of Cell Deaths exceeds the Number of Cell Divisions. In vivo this is when a Fever will Break or the Patient will very rapidly recover. You should try and be around so you can take some of the Credit, but donʼt ever forget that your Patientʼs Immune System did most of the Work.

Front 25

• Generation Time (also called Doubling Time)

Back 25

is the Interval between
successive Binary Fissions
- Generation Times are determined during Exponential Growth

Front 26

• Pure Culture

Back 26

A Population of Cells arising from a single Cell
Pure Culture Technique is the sine quo non (L= without this nothing)
of Microbiology.

Front 27

• Colony

Back 27

- A macroscopically visible Growth or Cluster of Microbes on a Solid Medium
• Each Colony represents a Pure Culture (or a Clone)
- Colony Morphology can sometimes aide in Identification of Bacteria Bacillus Colonies have a “Frosted Glass” Appearance. Streptomyces Colonies look like little Volcanoes (and smell like freshly plowed Soil).

Front 28

Culture Media
• Defined (Synthetic) Media

Back 28

- All Components are known
Major Elements (C, O, H, N, S, P) are usually present in Grams/Liter; Minor Elements (K, Ca, Mg, Fe) are usually present in Milligrams/Liter; and Trace Elements (Mn, Zn, Co, Mo, Ni, Cu) are usually present in Nanograms/Liter
Synthetic Media are Expensive and a Major Pain in the Butt to make.

Front 29

Culture Media
• Undefined (Complex or Enriched) Media

Back 29

- Actual Chemical Composition Unknown
- Contain Undefined Stuff like:
• Yeast Extract (Autolysed Yeast)
• Peptone (Protein Digest)
• Blood or Serum
• Beef Heart Extract
- Most Bacteria of Medical and Industrial Interest are quite Happy growing on Inexpensive, Easy-to-make Complex Media.

Front 30

Pure Culture Plating Techniques
• Streak Plate Isolation

Back 30

- Bacteria are transferred from Plate to Plate or Liquid to Plate using a Heat-Sterilized Metal Wire Loop (Inoculating Loop)
• The Loop also works well with Plates to Liquid or
Liquid to Plate transfers
- The Loop will hold ~0.01 ml (~10 µl) of Liquid
- Once Mastered, Streaking is Simple and Reliable

Front 31

Pure Culture Plating Techniques
• Spread Plate Isolation
-viable count method

Back 31

- Bacteria in a Suspension are pipetted onto a Plate and spread uniformly across the Plate with a sterile “Hockey Stick” Spreader or with a Sterile Swab.
• The Volume is generally ~0.1 ml (~100 µl)
- Spread Plate Isolation is best for more Uniform Results when dealing with Large Numbers of Samples

Front 32

Pure Culture Plating Techniques
• Pour Plate Isolation

Back 32

- Bacteria in a Suspension (0.1 to 1.0 ml/100 µl to 1000 µl) are added to approximately 20 ml of Molten Agar, which is then poured into a Petri Plate
- Allows Detection of both Aerobic Bacteria (on Top of the Agar) and Microaerophilic Bacteria (in the Agar)

Front 33

• Psychrophiles (G= cold loving
- Obligate Psychrophiles (Obligate ≈ “has to be”)

Back 33

• Cannot grow above 20°C
• Common in the Arctic, Antarctic, Mountains, and Ocean Floor
• Membranes have lots of Unsaturated Fatty Acids

Front 34

• Psychrophiles (G= cold loving)
- Facultative Psychrophiles (Facultative ≈ “prefers to be”)

Back 34

• Grow best at or below 20°C but can grow at higher
Temperatures
• Common in your Refrigerator

Front 35

• Mesophiles (G= middle loving)

Back 35

- Optimum 25°C to 40°C
- Most Bacteria
- Most Pathogens

Front 36

• Thermophiles (G= heat loving)
- Facultative Thermophiles

Back 36

• Grow best above 37°C but can grow at lower Temperatures
• Common in Compost Heaps and “cooler” Hot Springs

Front 37

• Thermophiles (G= heat loving
- Obligate Thermophiles

Back 37

• Cannot grow below 37°C
• Optimum 50° C to 60°C
• Common in Hot Water Lines (Heating Plants, Powerplants, Nuclear Reactors), Hot Springs
• Membranes with lots of Saturated Fatty Acids

Front 38

• Thermophiles (G= heat loving
- Hyperthermophiles

Back 38

• Optimum near 100°C
• Archaeobacteria near Deep Ocean Vents

Front 39

• Aerobic Bacteria
- Obligate Aerobes

Back 39

• Require O2 for Growth

Front 40

• Aerobic Bacteria
- Facultative Aerobes

Back 40

Do not require O2 but grow better in its Presence
They grow better under Aerobic Conditions than under Anaerobic
Conditions, but can grow under both Conditions

Front 41

• Aerobic Bacteria
- Microaerophiles

Back 41

• Require small Amounts of O2 but are damaged by higher
(Atmospheric) O2 Levels

Front 42

• Anaerobic Bacteria
- Aerotolerant Anaerobes

Back 42

• Ignore O2

Front 43

• Anaerobic Bacteria
- Facultative Anaerobes

Back 43

• Do not require O2 but grow better in its Absence
They grow better under Anaerobic Conditions than under Aerobic
Conditions, but can grow under both Conditions.

Front 44

• Anaerobic Bacteria
- Obligate Anaerobes

Back 44

• Do not tolerate the Presence of O2

Front 45

• Sterilize

Back 45

- The Elimination or Removal of all Living Entities
- An Absolute Term

Front 46

• Disinfect

Back 46

- The Elimination or Removal of contaminating Microbes
• Disinfectant
Disinfectants are usually Toxic Chemicals or Agents that eliminate or remove Disease-causing Microbes from an Inanimate Object .
• Antiseptic Antiseptics are usually Non-Toxic Chemicals or Agents that eliminate or remove Disease-causing Microbes from Skin or Tissue

Front 47

Heat

Back 47

Heat denatures Microbial Proteins

Front 48

Heat
• Direct Flame

Back 48

The oldest, most Direct Method for Sterilization

Front 49

Heat
• Dry Heat Sterilization

Back 49

- 160-170°C for 2 to 3 hours
• This is how most Labs sterilize Glassware

Front 50

Heat
• Boiling Water

Back 50

- Cannot eliminate Spores and so cannot Sterilize
- But conveniently Low-Tech

Front 51

Heat
• Autoclave (French = self-lock)

Back 51

- An Autoclave is basically an automatic Pressure Cooker
• 121°C 1.1 kg/cm3 15 Minutes
• 250°F 15 psi 15 Minutes (psi = pounds per square inch)
- This is how most Labs sterilize Media.
- This is how most Clinics sterilize Instruments.

Front 52

Filtration

Back 52

• Microbes are removed from Heat-sensitive Stuff via Microfiltration
- Pharmaceuticals, Cell Culture Media, Wine, Beer (“Cold Filtered”)

Front 53

Filtration
Membrane Filters

Back 53

• Made of Cellulose Acetate, Cellulose Nitrate, Polycarbonate

Front 54

Filtration
- High-Efficiency Particulate Air Filters (HEPA)

Back 54

• Air Filters for Sterile Hoods, Hospital Operating Rooms,
Industry Sterile Rooms, and Aircraft Air Conditioning Systems

Front 55

Irradiation

Back 55

• UV Light (~200 nm)
- Induces Thymine-Thymine Dimers in DNA
- Used for Lab Surfaces and small Instruments
• UV Light in Sunlight can disinfect Clothes dried Outside on a Clothesline
- Limited use because it doesnʼt penetrate Glass, Cloth, or

Front 56

Chemical Methods of Control
• Phenol and Phenolics (Phenol Derivatives)

Back 56

- Disrupt Cell Membranes and denature Proteins
- Examples: Lysol™, Listerine™, Hexacholorophene

Front 57

Chemical Methods of Control
• Halogens

Back 57

- Oxidize Cell Components
- Examples: Bleach (Sodium Hypochlorite), Iodine, Iodophor

Front 58

Chemical Methods of Control
• Alcohols

Back 58

- Disrupt Membranes and denature Proteins
- Examples: Ethanol, Isopropanol

Front 59

Chemical Methods of Control
• Soaps and Detergents

Back 59

- Disrupt Membranes and allow Microbes to be washed away
- Examples of Soaps: Ivory™, Castille™
- Examples of Detergents: Dial™, Bacdown™, GoJo™, Tide™