Notes on Microbiology Laboratory:

Isolation, Cultivation, Identification,

and Metabolism

Diana Verano M.S.

Pamantasan ng Lungsod ng Maynila
College of Nursing
 Microbial Taxonomy
• Taxonomy provides systematic way of naming,classifying and identifying
organisms.

Taxonomy includes:
 Classification
 nomenclature
 Identification

• Diagnostic medical microbiology involves classification and identification for

accuracy in diagnosis of infectious diseases.

• Classification of microorganisms even until this day is subjected to changes as

recent discoveries on homology of bacterial DNA and RNA elucidates
relatedness among taxonomic groups.
Homology: similarity

• Modern taxonomy:genotype+phenotype of microorganism.

[Link] characteristics –related to genetic makeup, the genes

 Phenotypic criteria of microorganisms

[Link] Morphology:
microbial growth patterns
observed on a specific
artificial media.

Example: size, texture,

pigmentation

totally dependent on the

media used.

Margin:refers to the edge of

the colony
Phenotypic criteria of microorganisms
Phenotypic criteria of microorganisms
2. Microscopic Morphology:
[Link] characteristics
[Link] requirements:
grow at different temperatures, presence or absence of
oxygen and other gases,pH levels or presence of ions and
salts like NaCl

[Link] requirements: ability to utilize various carbon

and nitrogen sources as substrates, under specific
conditions.

Physiological characterization: determines biochemical

character, and based on enzymatic activities
Phenotypic criteria of microorganisms
[Link] profiles:
 exhibition of a characteristic inherent resistance to specific
antibiotics, heavy metals, or toxins

[Link] properties: profile of microorganisms by various

serologic and immunologic methods to determine relatedness
among various microbial groups.

In actual laboratory set-ups

 Molds: usually identified through morphology (colony color,
age of colony, spore shape, hyphal septation)

 Yeast and bacteria:morphology, cultural growth, physiological

characterization (based on biochemical tests)
Phenotypic criteria of microorganisms
For Rapid tests:

 Rapid kit tests are used that contain different wells with
different media specified for many biochemical tests .
 For rapid profile Identification of Bacteria and yeasts
 microorganisms to be identified within 2-3 days.

Examples:
 API 20NE: for gram negative bacteria,non enteric (24-48hrs)
 API 20 E: enteric gram-negative bacteria (24-48hrs)
 API 20 AUX:sugar utilization for rapid yeast identification (24-
72 hrs)

 Note: API is a [Link] identification kits of different

brandnames can be used if available.
Nutrient Broth in test tube (24 hr old axenic culture) will be combined
with the liquid vial (part of test kit)
Combined liquid dispenses into small wells

Computer program assisted identification

Interpretation: color changes will generate numerical values

Type the number combination in computer program & microbial

identification appears
All the wells are MINITURIZED media or enzyme tests
 Phenotypic criteria of microorganisms

Rapid technology:
• Nowadays, Machines are employed to identify
microorganisms & antibiotic susceptibility testing ( Machine
automation Technology)
• with computer program
• - VITEK 2 ( machine brand name)
 Petrifilms ( Spread Plate Method)
• A drop of your sample ( diluted to 10 -1) is
dropped at the center of Petrifilm.
• Seal with the cover
• Use the spreader evenly
• Petrifilms come in different types
• ( yeast or TPC, or enterobacteriaceae)
• Colored colonies indicate identity if it is
enterobacteria

• Count all recognizable colonies

Detection of Microorganisms ( ATP)
Rapid technology: Bioluminometer
- Detects ATP ( Adenosine Triphosphate)
- 3M is just a brand name
- Swab Method
- Sterile swab is placed on a plastic enclosed
vessel with suspended sterile liquid)
- Validates hand washing of personnel &
cleaning of equiptments & surfaces
- Used in manufacturing plants ( food &
drugs) & hotels all over the world

Areas of swabbing
- Conver belts
- Delivery truck walls & floors
- Refrigerator storage Room surfaces
- Production areas
- Water facilities
 Detection of Microorganisms ( ATP
measurement)
Rapid technology: Bioluminometer
- More light, more ATP detected, the more
microorganisms remains on surface
- Acceptable value/range ( RLU): only
established by the company
- NO STANDARD COUNT FOR ALL INDUSTRIES
bec the technology is new
- Can be used in research for Hospital
contamination ( other countries)

- Example of acceptable range of food

companies: 0 - 150 RLU (relative light units)
If value is 151, the surface is not acceptable as
clean anymore

Instead of colonies forming units, this

equipment uses RLU
Laboratory Identification ( Classic Method)
 Bacterial cultivation

 Bacterial growth is required for identification and characterization

Medical point of view:

 Cultivation- process of growing microorganisms in culture by
taking microorganisms from infection sites (in-vivo environment)
by some means of specimen collection and growing to artificial
environment in the laboratory (in-vitro)
 Vitro came from the word vitrum meaning glass. So the word in-
vitro means in glass.

 Culture media contains nutrients suitable for bacterial growth

 Fastidious organisms- their needs are complex,require

exceptional media requirements
 Nonfastidious organisms- have basic nutritional
[Link] grow in most media
 Bacterial cultivation
 Colony: The bacterial population is considered derived from 1
bacterial cell

 Pure culture or axenic culture: All the colonies came from 1

single bacterial cell.

 Always with pure culture when performing biochemical tests.

Steps in Bacterial Cultivation

[Link]
[Link]
[Link]

Steps in Identification
 [Link] pure culture
 [Link]-stain procedure

 Gram Stain Morphology

Gram-positive Gram-positive Gram-negative Gram-negative

cocci bacilli bacilli cocci

Catalase reaction Growth in MacConkey Growth in Thayer-Martin

spores Agar Agar

+ + - +
Staphylococci Bacillus spp.
Enterobacteriaceae
-
Clostridium spp Haemophilus spp.
-
Streptococci
spp.
Actinomyces spp.
Brucella spp.
Legionella spp.
Or Enterococi Catalase reaction

Oxidase reaction Pathogenic Other

+ - Neisseria spp. Neisseria
spp.
Corynebacterium spp. Lactobacillus spp.
Listeria spp. Actinomyces spp. Pseudomonas + -
others Enterobacteriaceae
spp.

Identification scheme in Bacteria (Bailey`s and Scotts`s Diagnostic Microbiology)

 Isolation of [Link] from Sewage Water
[Link] :Streaked the Sewage water in EMB medium in 4 quadrants,store
in incubator in 24-48 hours at 37 °C.

Eosin Methylene Blue medium or EMB medium is a selective [Link]

Gram-negative bacteria grows in this medium.

-[Link] is distinguished by greenish metallic sheen

[Link]: Each quadrant should have 1 colony of greenish metallic

[Link] colonies are not found, streak again to another EMB plate

- If colonies are found,get the edge of the colony and transfer to broth or
agar [Link] for 24 hours in incubator.

[Link]: place the broth or slant in refrigerator for longer [Link] the
cotton plugs to absorb water to prevent contamination.
 Reminder
When performing identification tests, it should be:

• Identify if you are working with Axenic purified culture or mixed source
of sample ( canal water)
• Pure culture: Bacterial inoculum should be 18-24 hour old in Nutrient
Agar or Nutrient Broth
• Mixed source of sample: fresh sample, less than 24 hrs.
• Spores of 1-week-old pure axenic molds from PDA (potato dextrose
Agar).

• Test tubes whether it is Nutrient Broth,Nutrient Agar or Media for IMViC

contains 6mL of medium
 IMViC test for [Link] vs Enterobacter spp.
TESTS:[Link] [Link] red, [Link] Proskauer [Link]
[Link] results + + - -
Enterobacter spp. - - + +

Done in laboratory
[Link] test:
6ml SIM medium inoculated with organism (stab method)
After 24 hours: add kovac`s reagent
Positive test: red ring (without mixing)
[Link]-VP (Methyl Red, Voges-Proskauer)
MR test: get 1ml to a new tube, add 1-2 drops methyl red
Red color: acid production, yellow: negative
 IMViC test for [Link] vs Enterobacter spp.
Done in laboratory
[Link]-VP
6ml MRVP broth inoculated with organism:read after 24 hours
VP test: add 10 drops alpha-napthol and [Link] 15-20 drops
[Link] for 15-20 mins

Pink color to red: positive result

No change in color: negative result

[Link] utilization test

6mL Simmons citrate Agar (SCA) medium slants inoculated by stab streak
technique.
Blue: positive result(bromothymol blue indicator changes)
When citrate is utilized by bacteria, end product is alkaline, turning blue.
Green: no change
 Identification
• For spore formers, They are all [Link] it is aerobic it is Bacillus spp.

• If anaerobic, it is Clostridium spp.

• Phenotypic approach:biochemical tests and Gram staining are referred as

classical or conventional methods of identification of bacteria

• MacConkey Agar: is selective on Gram-negative [Link] isolated bacterial

colony on this medium is easily identified as Gram-negative.

• Wet mount preparation is also used for Identification.

• Colony characteristic if distinct on a particular organism is reliable.

• Example: growth of Campylobacter jejuni 42°C and Yersinia enterocolitica at 0°C

 Streptococcus spp.

- Gram-positive cocci occurring in chains or pairs with individual cells

- facultative anaerobes
-produce a small gray colony after 24 hour incubation at 35°C on sheep blood
agar.
-colonies grown under anaerobic conditions are larger than those grown
aerobically.
-catalase-negative

clinical identification of the streptococci has been guided by two factors:

[Link] serogrouping
• [Link] pattern on sheep blood agar.
• The Lancefield classification scheme is based on cell wall carbohydrate
constitution
• most important streptococci classified within the Lancefield scheme are
Group A S. pyogenes, Group B S. agalactiae, and GroupD
Streptococcus spp., not to be confused with Enterococcus spp., which also
posess the Group D antigen.
 Streptococcus spp.
• Three hemolytic patterns are commonly reported:
• [Link]-hemolytic streptococci cause an incomplete destruction of the red
cells within the sheep blood agar, producing a typical dark green discoloration
of the media surrounding or directly underneath the colony, reflecting the
presence of biliverdin and other heme compounds. The pneumococcus and
viridans-type streptococci are typically alpha-hemolytic, as are some
enterococci.
[Link]-hemolytic streptococci completely destroy the red cells in the sheep blood
agar, resulting in transparency of the agar. Streptolysin O is an oxygen-labile
hemolysin found in streptococci, especially in Group A S. pyogenes and, in
smaller amounts, in Group G and in some Group C streptococci. The oxygen-
stable streptolysin S is present in about 98% of Group A S. pyogenes isolates.
The zone of beta-hemolysis caused by Group A streptococci is therefore
enhanced if the culture is incubated anaerobically. Both Group A and Group B
streptococci are beta-hemolytic, as are several other streptococci of less clinical
relavance.
[Link]-hemolytic streptococci are most often found as commensals in respiratory
specimens, although they can occur in mixed cultures or as agents causing
UTI's. Also, Group D streptococci and enterococci can be alpha-, beta-, or non-
hemolytic.
 Phases of Growth Media

 [Link] broth
 [Link] agar

 Blood agar is biphasic medium (contains 2 phases).It has solid

agar base (autoclaved) + fresh liquid [Link] dispensing, the
2 components are mixed in flask.

 Broth medium- bacterial growth is indicated by turbidity

(cloudiness).Turbidity is due to light deflected by bacteria present
in the culture.

 More turbid means more bacterial growth (estimated at least 10 6

bacteria/mL for turbidity to be detected by naked eye)

 Solid media has agar

 Agar: common solidifying agent
 Phases of Growth Media
Test tubes can contain either
[Link]
[Link]
[Link]
[Link] slant (example: Simmon Citrate Agar)

 Slant :
 Example: Nutrient Agar Slant
 After autoclaving, Nutrient Agar is solidified at an angle in slanting
[Link] provide more surface area for streaking for aerobic
conditions.

 [Link] (SIM Medium) (media is in upright position)

 For anaerobic conditions
 Even Nutrient Agar can be used for anaerobic conditions for anaerobes

 Anaerobes in petri dishes are placed in anaerobic jars to create

anaerobic environment
Slant (streak method) broth

Butt slant in TSI Agar (triple sugar Iron)

 Greenish metallic
sheen of [Link] in EMB

Anaerobic jar
with gas pack
Butt or
deep test
tubes
Methods of Obtaining Pure cultures in Petri
plates
[Link] Plate method: (or multiple interrupted method/ quadrant streaking)
Objective: obtain isolated colonies, divide in 4 quadrants
Example: any sample to be analyzed (sewage,food etc)
-Also used as method to transfer pure culture from 1 petri plate to the next petri plate.

[Link] Plate Method: usually involves serial dilution of the sample with sterile water/peptone water
• Any sample to be analyzed: 100 dilution
• If you get 1g of sample+9ml sterile water and mixed: 10 -1dilution
• If you get 1 ml of 10-1dilution of sample+9ml sterile water and mixed: 10 -2dilution
• If you get 1 ml of 10-2dilution of sample+9ml sterile water and mixed: 10 -3dilution

• In the industry: 10-3 to 10-6dilution is used.1ml in the chosen dilution is placed on the sterile
plate+molten [Link] 24 hours, colonies will form and should be [Link] with the naked eye,
colonies are visible.

• Recent food & drug industrial methods: 10 -1 dilution but using a thin film of media: petrifilm
• (not media on petri plates), NOT USING POUR PLATE method. A variation of SPEAD PLATE
METHOD

• Example: any sample to be analyzed (sewage,food etc)

• Objective: obtain isolated colonies
 Methods of Obtaining Pure cultures
[Link] method:
Objective: obtain isolated colonies, antibiotic sensitivity tests
Example: From human body surfaces, also used for non living material, with
surfaces

[Link] plate method: needs a L-shaped rod spreader

• Small volumes of sample (up to 1ml of sample) spread entirely to the plate

• Will generate separate colonies like in pour plate provided that the sample
contains few colonies

• Industrial setting uses pour plate and spread plate when analysis of food
products or other industrial important products
 Techniques for Subculturing
microorganisms
If the organism is to be subcultured (transfer):
[Link] tube to petri plate: use streak plate method
[Link] dish to test tube: use the inoculating loop, obtain the edge of
1 chosen colony, transfer to test tube

 If 2 colonies or more are obtain upon subculturing,the test tube

will contain mixed culture.

 Mixed cultures will give inconsistent results and interpretation to

the identity of the organism.

[Link] plate to petri plate: streak method

 Techniques for Subculturing
microorganisms
If the organism is to be subcultured (transfer):
[Link] tube agar to test tube agar:
-in Nutrient Agar slants:use streak plate method (zigzag in tube)

5. test tube agar to test tube broth:

-use streak plate method (zigzag in tube) provided the broth
contains pure culture

Biochemical tests
[Link] method
-(for biochemical method in SIM medium)

[Link] streak (for SCA)

 Storage and Revival

Inoculation of Microorganisms like aerobic bacteria can be stored in

Nutrient Broth or Nutrient Agar slants, grown after 24 hours and
in incubator (37 °C) and placed in refrigerator (for prolonged
storage)

Revival of Cultures
-Cultures Stored from Nutrient Broth or Nutrient Agar or Lyophilized
Microorganisms (freeze-dried) should be inoculated in a Sterile
Nutrient [Link] turbidity.

-Turbidity indicates the microorganisms has been revived.

 Media classification

 According to Bailey`s and Scotts ,in bacteriology, there are 4

general categories of media (diagnostic microbiology)

[Link] media
 Enhances the growth of a particular pathogen that contains
specific nutrients.
 Example: buffered charcoal yeast extract agar: with L-cysteine,
specific for Legionella pneumophila (causes Legionaire`s disease)

 [Link] media – support growth of most non-fastidious

organisms.

 [Link] media- contains one or more agents that are inhibitory

to all organisms except the chosen organism.
 The media selects the bacterial growth
 Examples of inhibitory agents:bile salts, alcohol, acids antibiotics.
 Media classification

[Link] media – use some factors that allows colonies to exhibit

certain cultural characteristics that distinguishes them from other
bacteria on the same plate.
 Example: MacConkey Agar: differentiates lactose fermenters
gram-negative bacteria vs non-fermenters gram-negative
bacteria

 Fermenter: organism capable of fermentation

 Fermentation occurs when pyruvate in glycolysis is converted to
other products (Acids or alcohols) in the absence of oxygen.
 This is an alternative route for organisms to generate ATP, though
ATP generated is less compared to the usual ETC pathway.

 Some organisms can ferment using lactose as a substrate, while

others cant.
 Media classification

 In other references
 Simple medium:favors growth of all organisms
 Used for maintenance and cultivation

 Enrichment medium is different from enriched media

 Enriched medium: for fastidious organisms like Haemophilus
example: chocolate agar

 MacConkey Agar: is both differential and selective medium

 Selective:It will not allow gram-positive bacteria
 Differential: colonies are distinguishable

 Results in MacConkey Agar:

 Non lactose fermenters: colorless
 Lactose fermenters: pink to red
Examples of Medically important Media

[Link]-heart Infusion broth or Agar

 One of the components used in blood culture bottles.
Blood culture bottles is used for detection of bacteremia
( bloodstream infection)
 After obtaining blood sample from patient, blood is incubated with
blood culture [Link] blood cultures contain nutrient
broth+anticoagulant

[Link] Agar: Trypticase agar, Brucella agar, or beef heart infusion

agar+5% sheep blood (if no sheep blood available, human blood type
O is used)
Actual lab practice: agar base (sterilized) + blood, mix = dispense
[Link] Agar – almost the same with blood agar except the RBC is
lysed when added to the molten agar [Link] brown color. For
Neisseria gonorrhoeae (causes gonorrhea) and Haemophilus spp.
(infection of middle ear and respiratory tract)
 Lysed RBC releases hemin,hemoglobin, coenzyme NAD: utilization of
fastidious bacteria
 Sheep Blood Agar Hemolysis:
- activity of bacteria to lyse RBC by extracellular enzymes

 Beta hemolysis: complete clearing of RBC

 Alpha hemolysis: partial lysis of cells, green discoloration around the
colony
 Gamma or non hemolysis: no halo around RBC, no effect on RBC

Sheep blood agar – most specimen are inoculated especially for

fastidious organisms ( clinically significant)
- Medium contains agar base with protein source ( tryptones), soy bean
protein digest, sodium chloride, agar, 5% sheep blood
Examples of Medically important Media

4. MacConkey Agar
 Frequently used differential and selective media
 Contains crystal violet dye to inhibit growth of gram-positive
and fungi
 To isolate gram-negative bacteria
 pH indicator: neutral red to differentiate colonies
 Lactose fermenters: ([Link]) results in acid, lowering pH
medium,neutral red indicator gives the colony color
 Shigella: non lactose fermenter is colorless

Mannitol Salt Agar – peptone base with mannitol, phenol red

indicator. Salt concentration of 7.5% inhibits most bacteria.
- Selective for Staphylococci
Examples of Medically important Media

[Link] Enteric Agar: selective and differential

 Has bile salts and dye (bromothymol blue and acid fuschin) to
slow down growth of gram-negative bacilli non pathogens
 Allows Salmonella and Shigella growth
 Salmonella and Shigella colony: blue green color (like the
medium, no color change).They are not lactose
[Link] has black precipitate

 Ingredient:Ferric ammonium citrate, indicator for detection of

Sulfuric acid producing Salmonella for black precipitate.

 Non pathogens: orange or salmon colony color.

For lactose fermenters, produces acid and lowers medium
[Link] a change in the pH indicator bromothymol blue.
Examples of Medically important Media

[Link]-Martin Agar: for pathogenic Neisseria

 Has basal components of chocolatized blood+antibiotics
 Antibiotics: colicin (inhibits gram-positive bacteria),nystatin
(inhibits yeasts)
 Trimethoprim (inhibits Proteus spp.)

 Thayer-Martin Agar with antibiotics: for pathogenic Neisseria

meningitidis and [Link] only
 Common Media for Purification
• Once isolated colony is chosen, you could transfer in Nutrient Agar ( NA)
• Get a small portion of the colony and streak on NA, incubate for 24 – 48
hrs.

• For storage: from NA agar, get a small portion of colony thru inoculating
loop, transferred to Nutrient broth (NB) in tubes.

• Store NB tubes after 24 hr growth for bacteria.

 Identification
• Antibiotic Susceptibility tests:
• (swab method)
• Use McFarland Standard test tube,turbidity)
before swabbing of inoculum to petri dish
• ( ex. McFarland 0.5, 1, 2, 3,4)

• clinically significant gram-negative bacteria

have the potential to develop resistance to
vancomycin.

• Presence of zone of inhibition: indicative of

gram-positive bacteria

• Gram-negative bacteria:susceptible to
colistin or polymyxin, gram-positive bacteria
are resistant.
 Enzyme-based Tests
• Designed to measure the presence of 1 specific enzyme.

• Single enzyme tests: for presence of 1 enzyme

 Catalase test: for gram – positive bacteria
 Oxidase test: for gram – negative bacteria

Catalase test
 Test for presence of catalase
 Catalase: enzyme for release of water and oxygen from hydrogen
peroxide
 H2O2+ catalase = H2O+ O2

 Positive test interpretation: rapid bubbles (effervescence) if bacterial

culture +hydrogen peroxide solution mixed
 Negative test interpretation: no bubbles

 Staphylococci and Listeria monocytogenes: catalase positive

 Enzyme-based Tests
Oxidase test: test for presence of cytochrome oxidase, obligate
aerobes have this enzyme.

Obligate aerobes: organisms that need oxygen during respiration

 Cytochrome oxidase-participates on Aerobic respiration on ETC

(electron transport chain) and nitrate metabolic pathway

 Use wooden sticks in obtaining [Link] wire of the loop may

have false positive result.

 Positive result interpretation: purple color (only up to10 seconds)

 Negative result: no [Link] after 30 seconds due to oxygen

exposure of air,the color will also be purple.
 Enzyme-based Tests
Oxidase test:
 Enterobacteriaceae family members: oxidase negative
 Example: [Link], Enterobacter [Link] are enteric
(found in human intestine and fecal matter).They are also
facultative anaerobes.

 Facultative anaerobe: grows with or without oxygen

 If Oxygen present: more ATP produced
 If oxygen absent: glycolysis proceeds to fermentation.
 Sugars present on the medium is converted to organic acids
or alcohol (this depends on the organism)

 Oxidase is absent on facultative anaerobes.

 Enzyme-based Tests
Oxidase test:
 Oxidase positive organisms: Pseudomonas aeruginosa,
Burkholderia cepacia, Ralstonia and Neisseria spp.

 Pseudomonas spp. are biochemically versatile

[Link] and Ralstonia before was identified
and used the Genus [Link] is because they
have common characterisitcs:oxidase positive, and
versatility to utilize different sugars

 Only on the molecular identification proved that these

organism be separated on 3 different genera.

 Recent rapid kits can distinguish the 3 genera as

[Link] 3 genera are all found in soil.
 Enzyme-based Tests
Indole test:test for the presence of indole

 Indole is not an enzyme but a metabolic product of tryptophan

 Tryptophan is an amino [Link] it is degraded:

tryptophanase
 Tryptophan pyruvic acid,ammonia and indole

Tryptophanase: enzyme

 Interpretation:
Positive: [Link]
Indole+aldehyde (indicator): blue color
Negative: Enterobacter cloacae
Colorless
 Physiological Characteristics
• Microorganisms grow only when conditions are met to favor their
metabolic process of growth.

• Microorganisms have different physical and chemical requirements

to favor their growth.

[Link] requirements
Organisms are classified as
• [Link]: use UV (inorganic substance) to generate ATP
• Example: plants, algae (have chlorophyll)
• [Link]: use complex organic compounds to generate ATP
• Example: humans, fungi
 Physiological Characteristics
• Microorganisms grow only when conditions are met to favor their
metabolic process of growth.

• Microorganisms have different physical and chemical requirements

to favor their growth.

[Link] requirements
Organisms are classified as
• [Link]: use UV (inorganic substance) to generate ATP
• Example: plants, algae (have chlorophyll)
• [Link]: use complex organic compounds to generate ATP
• Example: humans, fungi
 Physiological Characteristics
[Link] requirement
Organisms classified according to oxygen requirements:
[Link]: grows in the presence of oxygen
Strictly or obligate aerobe: needs oxygen as final electron acceptor
Ex: humans, Bacillus subtilis.
Microaerophile: needs only small amount of oxygen
Example: Yersinia pestis (plague) Campylobacter jejuni
[Link]:oxygen is lethal
[Link] anaerobe: can grow with or without oxygen
• If oxygen present: produce more ATP, oxygen as final electron acceptor
• If no oxygen: fermentation occurs (ex: beer and wine)
• Pyruvate is converted to alcohol or other organic acids as final electron acceptor
• Ex: [Link], Saccharomyces cerevisiae (yeasts)
 Some microorganisms can ferment
food,some can`t.

 Fermenter: organism capable of

fermentation

 Fermentation : when pyruvate in glycolysis

is converted to other products (Acids or
alcohols) in the absence of oxygen.

 an alternative route for organisms to

generate ATP, though ATP generated is
less compared to the usual ETC pathway.

 Some organisms can ferment using lactose

as a substrate, while others cant.
 Physiological Characteristics
[Link] requirement
[Link]:grows in the absence of oxygen
Obligate anaerobe-lives in the absence of oxygen
Example: Clostridium tetani
Facultative anaerobe: can grow with or without oxygen
• If oxygen present: produce more ATP, oxygen as final electron acceptor
• If no oxygen: fermentation occurs (ex: beer and wine)
• Pyruvate is converted to alcohol or other organic acids as final electron
acceptor
• Ex: [Link], Saccharomyces cerevisiae (yeasts)
Aerotolerant: doesn’t grow well but survives in the absence of oxygen
Example: Clostridium tertium
 Toxic forms of Oxygen
1. Singlet Oxgen 1O2
• Organisms that are
airborne,phototrophs and other
microorganisms encounter singlet
oxygen,produce carotenoids

[Link] H2O2– most reactive, can

damage cell parts

Enzymes:
Catalase: forms peroxide to water and
oxygen

Peroxidase: forms peroxide to water only

 Toxic forms of Oxygen

[Link] O2 –

SOD or enzyme superoxide dismutase generates hydrogen

peroxide and oxygen

SOD works with catalase

 Physiological Characteristics
[Link] requirement
• Optimum temperature: best suited for growth
• Minimum temperature: lowest temp. for growth
• Maximum temperature: highest temp. for growth

• Psychrophilic: cold loving bacteria 10-20 °C

• Mesophilic: grows best in moderate temperature, pathogens
• 5 °C :minimum temp.
• 30-37 °C :optimum
• 43 °C :maximum

• Thermophilic: grows at higher temperature

• optimum:50-55 °C
Physiological Characteristics

1: Obligate aerobi c bacteria

2: Obligate anaerobic bacteria gather at the bottom to avoid oxygen.
3: Facultative anaerobe bacteria
4: Microaerophiles
5:aerotolerant bacteria
 Physiological Characteristics
[Link] (hydrogen ion concentration)
• Most pathogens require neutral or slightly alkaline pH
• Acidophilic: requires acidic environment
• Ex: Lactobacillus,(in vaginal area) protects women from infection of other
microorganisms that is also found in female genital parts.
• Basophilic: requires basic medium example: Vibrio cholerae (causes cholera)

[Link] activity: availability of water [Link] water molecules are trapped

with solutes, it is not available.
• For yeasts and pathogenic bacteria, more water molecules available favors
faster [Link] causes rapid spoilage in food and other industrial products
• Molds has less water activity [Link] can grow on dry
[Link] attacks food and products with highly concentrated solutes.
 Physiological Characteristics
[Link] content

Halophiles: requires high solute concentration of solutes

ex: Vibrio parahemolyticus found in sea water and uncooked
seafoods.