INTRODUCTION TO MICROBIOLOGY, INFECTION CONTROL AND LAB SAFETY

MED-21A- Mr. Ariel Salvacion

BACTERIAL PHYSIOLOGY,
METABOLISM, GENETICS, GROWTH
AND NUTRITION, AND BACTERIAL
IDENTIFICATION
iv. Inclusions
• Metachromatic Granules – represents reserves of
polyphosphates used in the synthesis of ATP.
• Polysaccharide Granules - consist of glycogen and
starch granules
v. Spores
• highly refractile resting cells that are highly durable
and dehydrated with thick walls

B. CELL ENVELOPE
BACTERIAL PHYSIOLOGY
i. Plasma Membrane (Cell
ii. Cell Wall
PROKARYOTIC CELL STRUCTURES
iii. Periplasmic space
• Prokaryotic Cell Components iv. Outer Membrane
A. Cytoplasmic Structures
B. Cell Envelope
B. Cell Envelope Structures
i. Plasma Membrane (Cell Membrane)
C. Surface Polymers or Appendages
• A phospholipid bilayer with embedded proteins that
envelop the cytoplasm.

A. CYTOPLASMIC STRUCTURES
i. nuclear Area (nucleoid) ii. Cell Wall (Peptidoglycan or murein layer)
ii. plasmid • Maintains the shape of the cell and protects cell
iii. ribosomes from osmotic pressure
iv. inclusions • Repeating disaccharide attached by
v. Endospore polypeptides
A. CYTOPLASMIC STRUCTURES
i. Nuclear Area (Nucleoid)
• single circular chromosome attached to a
mesosome
ii. Plasmids
• small circular, dsDNA molecule iii. Periplasmic space
• antibiotic resistance
• Between cell membrane and cell wall.
iii. Ribosomes
• Consist of gel like matrix containing nutrient
• site of protein synthesis
binding nutrients
• consists of RNA and protein
• Contains enzymes for degrading and detoxifying
macromolecules

Montalban, Kimberly N. & Barredo, Althea Kristine

BSMT3 MED21A
INTRODUCTION TO MICROBIOLOGY, INFECTION CONTROL AND LAB SAFETY
MED-21A- Mr. Ariel Salvacion
ii. Flagella
A. CYTOPLASMIC STRUCTURES • Exterior protein filaments that rotate and cause
iv. outer membrane bacteria to be motile
• Cells initial barrier (certain antibiotics and • Four arrangements of flagella
evasion of phagocytes) a. Monotrichous - a single polar flagellum
• Composed of Lipopolysaccharides (LPS), b. Lophotrichous - two or more flagella at
lipoproteins and phospholipids. one pole of the cell.
• Contains Porins c. Amphitrichous - single/tuft of flagella at
- Water-filled structures that control each end of the cell.
the passage of nutrients and solutes d. Peritrichous - distributed over the
entire cell.
iii. Fimbriae and Pili
CELL ENVELOPE
• Hairlike appendages that are shorter, straighter
CHARACTERISTIC GRAM + GRAM -
and thinner than flagella.
Peptidoglycan Thick Absent a. Fimbriae (sing. fimbria) - also called common
layer (multilayere) pili, can occur at the poles or can be evenly
Teichoic acid Present in many Absent distributed from few to several hundred.
b. Pili (pilus) - also called sex (conjugation) pili, are
Periplasmic space Absent Present hollow protein tubes, longer than fimbria and
number is 1 or 2 per cell.
Outer Membrane Absent Present •FUNCTION:
a. Fimbriae- for adherence of cells to one another
and to environmental surfaces.
LPS content Virtually none High
b. Pili (pilus) – to join bacterial cell in preparation
Lipid and Protein Low High (due to of DNA transfer from one cell to another.
outer
membrane)
BACTERIAL METABOLISM
C. Surface Polymers and Appendages
• Various biochemical pathways exist for substrate
i. Glucocalyx (slime layer and Capsule)
breakdown in the microbial world, and the
ii. Flagella
particular pathway used determines the end
iii. Pili
product and final pH of the medium.
iv. Axial Filaments
• Diagnostic Schemes
C. Surface Polymers and Appendages 1. Utilization of various substrates as a carbon
i. Glucocalyx (slime layer and Capsule) source
• General substances that surround cells. 2. Production of specific end products from various
• Gelatinous polymer of polysaccharide, substrates
polypeptide, or both. 3. Production of an acid or alkaline pH in the test
a) Capsule - substance is organized and firmly medium
attached to cell wall. I. Fermentation and Respiration
b) Slime Layer - unorganized and loosely attached A. Fermentation - anaerobic process
to the cell wall. carried out by both obligate and
• Functions facultative anaerobes
- Prevents phagocytosis - end products: lactate,
- Attachment to various surfaces in its natural ethanol, butyrate,
environment - Used for MRVP
B. anthracis, S.pneumoniae , K. pneumoniae
c) S. pneumoniae
d) K. pneumoniae
Montalban, Kimberly N. & Barredo, Althea Kristine
BSMT3 MED21A
INTRODUCTION TO MICROBIOLOGY, INFECTION CONTROL AND LAB SAFETY
MED-21A- Mr. Ariel Salvacion
B. Respiration - efficient energy-generating process BACTERIAL GENETICS
in which molecular oxygen is the final electron
acceptor ➢ MECHANISMS OF GENE TRANSFER
• Obligate aerobes and facultative anaerobes • Transformation - uptake and
incorporation of naked DNA into a
➢ Glucose to Pyruvic Acid bacterial cell
I. Embdem-Meyerhof-Parnas (EM) Glycolytic o S. pneumoniae, N. gonorrhoeae, H.
Pathway influenzae
II. Pentose Phosphate Pathway • Transduction - transfer of bacterial genes by
III. Entner-Doidoroff Pathway a bacteriophage
❖ ANAEROBIC UTILIZATION OF PYRUVIC ACID o C. diphtheriae
• Alcoholic fermentation - major end product is ethanol • Conjugation - transfer of genetic material
o Used by yeasts from a donor bacterial strain to a recipient
• Homolactic fermentation - end product is almost strain.
exclusively lactic acid
o Streptococcus spp., Lactobacillus spp.
• Heterolactic fermentation - end products are lactic
acid, CO2, alcohols, formic acid, acetic acid
• Propionic acid fermentation - end product is propionic
acid.
o Carried by Propionicbacterium acnes
• Mixed acid fermentation - lactic, acetic, succinic,
formic acids BACTERIAL GROWTH AND NUTRITION
o Escherichia spp., Salmonella spp., Shigella spp.
• Butanediol fermentation - end products are acetoin I. Nutritional Requirement
and 2,3-butanediol ✓ Autotrophic
o Klebsiella spp., Enterobacter spp., Serratia spp. ✓ Phototrophs
✓ Chemotrophs
➢ CARBOHYDRATE UTILIZATION AND LACTOSE
✓ Lithotrophs
FERMENTATION ✓ Heterotrophs/ Organotrophs
• Ability of m.o. to use various sugars for growth is ✓ Saprophytes
an integral part of most diagnostic identification ✓ Parasites
schemes II. Oxygen Requirement
• Fermentation of sugar is usually detected by acid ✓ Obligate aerobes - requires oxygen
production and a concomitant change of color for growth.
• Due to pH indicator present in the culture medium ✓ Obligate anaerobes - grows in strict
• For Enterobacteriacene: determination of the m.o. absence of oxygen.
ability to ferment lactose ✓ Facultative anaerobes/ Facultative
o Lactose fermenters, lactose non-fermenters, aerobes - can growth without
late lactose fermenters oxygen, but upon will utilize upon
its presence.
✓ Microaerophiles - requires small
amount of oxygen.
✓ Aerotolerant - can grow without
oxygen but can survive in the
presence of it.

Montalban, Kimberly N. & Barredo, Althea Kristine

BSMT3 MED21A
INTRODUCTION TO MICROBIOLOGY, INFECTION CONTROL AND LAB SAFETY
MED-21A- Mr. Ariel Salvacion
III. CO2 Requirement 4. Death or Decline Phase
✓ Capnophiles - requires CO2 to grow ▪ Period of cessation of bacterial growth as the
a. Neiserria gonorrheae number of dead cells exceeds the living
b. Haemophilus influenzae microorganisms
c. Brucella abortus
d. Streptococcus pneumoniae
e. Mycobacteria spp.
IV. Temperature requirement
i. Psychrophilic/cryophilic- low temperature
(10-20C)
ii. Mesophilic - where most pathogens grow
(30-44C or 20-45C)
iii. Thermophilic - heat loving (50-60C)
iv. Hyperthermophilic- (80-113C) BACTERIAL GROWTH CURVE
V. pH requirement
o Acidophilic - requires acid med (pH 0 - 5.5) ➢ CULTURE MEDIA
▪ Lactobacilli ▪ any substance that contains the nutritional
o Basophilic - (pH 8.5 - 11.5) requirements needed for bacterial growth
▪ Vibrio cholerae ▪ CULTURE - group of organisms obtained in a
o Neutrophilic - optimum pH 6.5 - 7.5 of most media
bacteria o 3 TYPES OF CULTURE
❖ Other Requirements ✓ Pure Culture
VI. Hemidophilic - moisture content ✓ Mixed Culture
VII. Halophilic - salt concentration ✓ Stock Culture
VIII. Osmophilic - high osmotic pressure ❖ CLASSIFICATIONS OF CULTURE MEDIA
➢ CONSISTENCY
BACTERIAL GROWTH CURVE ✓ Liquid Medium - does not contain any
amount of agar, allows growth of aerobes,
Stages of Bacterial Growth Curve anaerobes, and facultative anaerobes
✓ Generation time of bacteria in a culture ✓ Semi-solid medium - contains 0.5% - 1%
varies according to their cellular properties agar, used to observe bacterial motility and
✓ It takes 20 mins for fast growing bacterium detect indole and sulfide production
(E. coli) or 24 hours for slow growing ✓ Solid culture medium - 2% to 3% agar
bacteria. ➢ COMPOSITION
✓ Synthetic culture medium
Stages of Bacterial Growth Curve
- All components are known
1. Lag Phase or Period of Rejuvenescence
- Research purposes
▪ Period when there is no cell division, start of
✓ Non-synthetic culture medium
biosynthesis, adjustment phase to a new
- Some of the substances are
environment
unknown
2. Log or Exponential Phase
- Isolation of medically
▪ Period when microorganisms are actively growing
significant bacteria
and dividing, phase where m.o. are utilized in
✓ Tissue culture medium
physiological, biochemical, and antimicrobial
- For obligate intracellular
testing
bacteria
3. Stationary/Plateau Phase
➢ MEDIUM DISPENSED
Balance between cell division and dying organisms, but
✓ Plated medium
number of viable organisms are constant
✓ Tube medium
a. Liquid

Montalban, Kimberly N. & Barredo, Althea Kristine

BSMT3 MED21A
INTRODUCTION TO MICROBIOLOGY, INFECTION CONTROL AND LAB SAFETY
MED-21A- Mr. Ariel Salvacion
b. Slant
• Inoculation of Culture Media TECHNIQUES
c. Butt
➢ Liquid medium
d. Butt and slant
➢ Plated medium
USE ➢ Butt medium – stabbing
I. Simple media - routine in the lab, w/o ➢ Slant medium – streaking
additional supplements ➢ Butt/slant - stab the butt portion then streak the
II. Enrichment medium (liquid-type media) slant medium
- enhances the propagation
III. Enriched medium - with additional
supplements (blood, vitamins,
IV. Differential medium - distinguishes
organisms growing together by their
differences in cultural characteristics
V. Selective medium - promotes the growth
of desirable organisms but at the same
time inhibiting the growth of others • METHODS OF STREAKING For Plated Media
VI. Special/Selective culture medium ➢ Radial streak method
➢ Overlap streak method - for sensitivity
testing
V.SELECTIVE MEDIUM USE ➢ Multiple streak method
✓ Substances that inhibit gram positive ➢ Interrupted streak method
organisms ➢ Multiple interrupted streak method
• Gentian violet
• Sodium desoxycholate and other bile salt
✓ Substances that prevent swarming of Proteus
spp.
• Alcohol
• Chloral hydrate
✓ Substances that inhibit gram negative
organisms
• Potassium tellurite
• Sodium azide
• Neomycin and sodium azide

VI.SPECIAL/SELECTIVE CULTURE MEDIUM OBTAINING PURE ISOLATED COLONIES

✓ Lowenstein-Jensen/7H-9 broth- M. tb
✓ Thayer-Martin - Neisseria group
✓ Macvtrode agar- L. monocytogenes
✓ Fletcher medium - Leptospira
✓ W medium - Brucella
✓ Bordet-Gengou Agar - B. pertussis
✓ Mannitol Salt Agar - Staphylococci
✓ Saboraud Dextrose Agar – fungi
✓ Loeffler’s Medium/ Tinsdale/Tellurite Media-
C.diphtheriae
✓ Streak plate method
✓ Pour plate method - used to determine the
appropriate number of viable microorganisms in
a liquid medium
• Reported as number of colonies per mm of
inoculum
✓ Using selective medium/containing antibiotics
✓ Animal inoculation test

Montalban, Kimberly N. & Barredo, Althea Kristine

BSMT3 MED21A
INTRODUCTION TO MICROBIOLOGY, INFECTION CONTROL AND LAB SAFETY
MED-21A- Mr. Ariel Salvacion
➢ ANAEROBIC CULTURE
✓ Culture media should be protected from oxygen
exposure, freshly prepared, and should be held
in anaerobic condition until needed.
✓ Recommended methods: Thioglycolate broth &
anaerobic jar
✓ Use of anaerobic chamber with vacuum pump
for processing of samples and storage of culture
media prior to inoculation
❖ ANAEROBIC CHAMBER: Sealed Glove Box and
Gloveless Chamber
1. Nitrogen gas - acts as a filler for the remaining
percentage of the anaerobic atmosphere
2. Hydrogen and carbon dioxide gas - facilitates the
growth and isolation of anaerobes
3. Palladium catalyst pellets – remove residual oxygen
from the chamber
4. Silica gel (desiccant) - absorbs the water that is
formed when hydrogen combines with free oxygen
5. Methylene blue/reazurin- oxygen reduction
incubator
➢ INCUBATION
- CO2 incubator is not
available, used candle gar or
Gaspak jar with CO2
generator
➢ REPORTS
✓ Save culture until satisfactory growth is
obtained and final diagnosis is made.
✓ Some cultures made be held for several weeks
before final report is made
o Blood culture - 7-10 days
o Brucella and M. tb - 4 weeks
o L. monocytogenes - 4 months
✓ All media except thioglycolate broth should be
stored in the refrigerator .
✓ The number of media stored should not exceed
2-3-week supply.
✓ All media must be checked before use.
Montalban, Kimberly N. & Barredo, Althea Kristine
BSMT3 MED21A
COLONIAL GROWTH
• COLONIES - groups of bacteria forming on solid
media as a result of division of one or a few
organisms
• TYPES OF COLONIES:
1. S or smooth colonies - uniform texture &
homogeneity, easily emulsified in NSS, virulent
organisms
2. M or mucoid colonies - associated with capsulated
and virulent m.o., slimy/watery
3. R or rough colonies - granulated in appearance,
hard to emulsify in NSS
BACTERIAL IDENTIFICATION
➢ Conventional Methods
- Biochemical characterization serves as the
fundamental source of information for ID of
most bacteria after microscopy.
• Motility test - wet mount and hanging drop preparation
• Staining - Gram staining, acid-fast staining, structural
staining
• Manual biochemical test - carbohydrate fermentation
(LIA), catalase and coagulase tests
• Use of routing and selective media
➢ Analytical Profile Index
- Consists of plastic strips and microtubes that
contain dehydrated biochemical substrates
• Commonly used for gram (-) enteric bacteria
• Same principle with biochemical manual tubed method
• Principle: Biochemical substrates (pH-based substrates)
are inoculated with pure culture suspended in sterile
physiologic saline and incubated for 18-24 hrs at 35C, some
reagents may be added after incubation.
INTRODUCTION TO MICROBIOLOGY, INFECTION CONTROL AND LAB SAFETY
MED-21A- Mr. Ariel Salvacion
➢ Automated Method for ID of Bacteria
• Patterns of microbial growth are compared with
the test organism using a computer software
• Growth is detected through colorimetric,
fluorometric, or turbidimetric analysis
• Used for various m.o.
• Principle: Pure bacterial isolate during
incubation at 35C for 16-24hrs utilized the
carbohydrate in the reagents, thus producing acid
or alkaline end products as early as 2 hours.
• BD Phoenix System, Vitek System
MICROSCOPY
MICROSCOPY
• Fundamental method used for both the
detection and characterization of bacteria
• Microscope - vital in magnifying m.o.
❖ Remember:
▪ Unit of measurement in determining
bacterial size is micrometer (um)
▪ Ocular micrometer is located within the
eyepiece
▪ Refractive index - measure of the
relative velocity at which light passes
through a material or specimen
▪ Total magnification - product of the lens
and the objectives that are used.
Montalban, Kimberly N. & Barredo, Althea Kristine
BSMT3 MED21A
TYPES OF MICROSCOPES
1. Light Microscope - visible light passes through the
specimen then through the lenses that reflect light
a. Brightfield microscope - most commonly used
microscope, forms a dark image against a brighter
background, can distinguish between 2 dots that are 0.2
um apart
b. Phase contrast microscope - permits detailed
examination of internal structures in living organisms,
fungi, staining not included
c. Fluorescence microscope - involves excitation of
fluorochromes (dye) using light
d. Darkfield microscope - uses a dark field condenser
that blocks light, used to observe spirochetes.
2. Electron Microscope - utilizes electrons instead of
light
- Has electromagnetic fields instead of lenses to form
images
- Presence of built-in camera to capture images of cells
in black-and-white transmission
a. Transmission Electron Microscope (TEM)
• Allows visualization of the internal structures of cells
• Resolving power: 0.2 nm, magnification: 150, 000x to
10M x
• Used to examine very thin specimens and m.o.
b. Scanning Electron Microscope (SEM)
• Scans the surface of the cells or specimens
• Resolving power: 200 nm, magnification: 20 to
10,000x
•Specimen is positioned at the bottom of the column
3. Digital Microscopy
• A technology that utilizes the science of staining like
the gram reaction
• Capture cellular images through web-based interface
• Uses an automated microscope and the interface to
present images on screen or computer monitor
INTRODUCTION TO MICROBIOLOGY, INFECTION CONTROL AND LAB SAFETY
MED-21A- Mr. Ariel Salvacion
• All spiral organisms are reported as gram
negative.

STAINING TECHNIQUES

▪ Types of Stains
✓ Simple Stains
✓ Indirect, Relief, or Negative Stains
✓ Special Stains - Capsular Stain, Cell Wall Stain,
Metachromatic Granules, Flagellar
✓ Differential Stain

❖ GENERAL RULES FOR GRAM STAINING

• All cocci are gram positive except
Neisseria,Branhamella, Moraxella, Velionella.
• All bacilli are gram negative except
Mycobacteria, Clostridium, Corynebacterium,
Bacillus,Erysipelothrix, Listeria, Lactobacillus.
• Higher forms of organisms (Actinomyces,
Streptomyces, yeasts, and molds) are gram
positive.

Montalban, Kimberly N. & Barredo, Althea Kristine

BSMT3 MED21A