BIO

351 Practical Review

Lab 1 BRIGHTFIELD MICROSCOPY; SURVEY OF THE MICROBIAL WORLD; AIR PLATES

Shapes of Bacteria
o Cocci ROUND
o Bacillus ROD
o Spiral (Helical)
Arrangement
o Strep CHAIN
o Staph CLUSTERS
Protozoa

Paramecium
Molds & Yeasts

Penicillium conidia

Budding Yeast
Saccharomyces cerevisiae

Bacteria

Lab 2 SMEAR PREPARATION; SIMPLE STAINING; GRAM STAINING

Preparation of Bacterial Smears
o Bacterial Staining
Stain: an organic compound containing a benzene ring, a chromophore, and an
auxochrome group
Acidic stains: negative charge
Basic stains: cationic; frequently used to view shape and arrangement
o Positively charged ions attract to negative charge
Simple staining: bacterial smear is stained with a single reagent
o Purpose to elucidate the morphology and arrangement of bacterial cells
Examples methylene blue, crystal violet, and carbol fuchsia
Gram Stain

o Differential staining: requires the use of at least 3 chemical reagents that are applied sequentially
to a heat-fixed smear
Purpose Classification and differentiation of microorganisms
Primary stain first reagent
Imparts its color to all cells
Secondary stain decolorizing agent used to remove/retain primary stain
Tertiary stain counterstain; has a contrast color to the primary stain
If the primary stain isnt washed out, the counterstain cant be absorbed and the
cell will retain the PRIMARY STAIN
Examples Gram stain divides bacterial cells into gram-positive & gram-negative
Primary stain crystal violet (Huckers) PURPLE
Mordant (substance that increases the cells affinity for a stain by binding to the
primary stain) Grams iodine intensifies PURPLE (crystal-violet complex)
Decolorizing agent ethyl alcohol 95% (protein-dehydrating agent & lipid solvent)
G+ = PURPLE G- = COLORLESS
Counterstain Safranin Stains decolorized cells PINK (ONLY GRAM-NEGATIVE
CELLS UNDERGO DECOLORIZATION)
o G+ = PURPLE B. megaterium, S. aureus
o G- = PINK P. aeruginosa , P. vulgaris

Lab 3 & 4 PURE CULTURE TECHNIQUES & BACTERIAL POPULATION COUNTS

Techniques for Isolation of Pure Cultures

o Pure cultures cultures that contain only one type of organism and are suitable for the study of
their cultural, morphological, and biochemical properties
Techniques used to isolate pure cultures
Streak-plate a dilution technique that involves spreading a loopful of culture
over the surface of an agar plate (Manual)
o E.g. quadrant streaking
Spread-plate requires that a previously diluted mixture of microorganisms be
used (Microbes previously diluted)
Bacterial Growth Curve
o Purpose
Used to delineate stages of the growth cycle
Facilitates measurement of cell numbers and the rate of growth of a particular organism
o Stages of Bacterial Growth Curve
Lag Phase
Cells are adjusting to their new environment
Cellular metabolism is accelerated
Cells are increasing in SIZE
No cell division = NO growth in numbers
Log Phase
Optimum nutrition and physical conditions
Rapid exponential growth in population that doubles until the maximum number
of cells is reached
Generation time: Time required for a microbial population to double
Stationary Phase

Number of cells undergoing cell division = Number of cells dying

No further increase in cell number
Cell population is maintained at its maximum
Caused by depletion of essential metabolites and accumulation of toxic
acidic/alkaline end products in the medium
Death Phase
Continuing depletion of nutrients and buildup of metabolic wastes
Microorganisms begin to die at a rapid and uniform rate

Lab 5 & 6 TEMPERATURE: EFFECTS ON GROWTH; OSMOTIC PRESSURE & BACTERIAL
GROWTH; UV LIGHT: LETHAL EFFECTS; BACTERIOLOGICAL EXAMINATION OF WATER
(MULTIPLE TUBE METHODS)

Cultivation of Microorganisms
o Nutritional Needs
Carbon
Autotrophs obtain inorganic form of carbon (CO2)
Heterotrophs obtain organic form of carbon (glucose)
Nitrogen
Important for proteins and nucleic acids
Nonmetallic elements
Sulfur
o Found in amino acids
Organic form sulfur containing amino acids
Inorganic form sulfates; elementary sulfur
Phosphorus
o Found in nucleic acids of DNA/RNA
o Synthesis of ATP
Metallic elements zinc, magnesium, sodium, potassium
Osmoregulation
Electron transport chain (ETC)
Vitamins
Organic compounds required for cell growth
Coenzymes
Pathways to synthesize vitamins (E. coli)
Water ALL require water
Energy
Phototrophs obtain energy from the sun
Chemotrophs obtain energy from oxidation reactions
o Physical Factors
Temperature
pH
Gaseous Requirement
Physical Factors Temperature
o Minimum growth temperature lowest temperature growth can occur
o Maximum growth temperature highest temperature growth can occur
o Optimum growth temperature temperature at which rate of growth is rapid

o Psychrophiles -5 20C
o Mesophiles 20 45C
35 40C is optimal temperature for human host
o Thermophiles 35C+
Facultative thermophiles grow at 37C; optimum is 45 60 C
Obligate thermophiles grow ONLY above 50C; optimum 60C
Environmental Osmotic Pressure (water always moves from HIGH LOW)
o Hypertonic high osmotic pressure, high solute concentration
Net movement of water OUT cell shrivels
o Hypotonic low osmotic pressure, low solute concentration
Net movement of water IN cell LYSIS
o Isotonic equal concentration of solute and water
NO osmosis
o 0.5 3% NaCl can survive
o 10 15% - microogranisms die
o Halophile can survive in high salt concentrations (E.g. S. aureus)

Lab 7 & 8 MICROBIAL GROWTH CONTROL; ANTISEPTICS; ANTIMICROBIC SENSITIVITY

TESTING

Physical & Chemical Agents for the Control of Microbial Growth
o Antiseptics chemical substances used on living tissue that kill or inhibit growth of vegetative
microbial forms
Iodine
o Disinfectants chemical substances that kill or inhibit the growth of vegetative microbial forms
on NONLIVING material
Bleach
Chemical Agents of Control: Disinfectants & Antiseptics
o Chemical substances used to prevent contamination and infection
Efficiency is influenced by
Concentration
Length of Exposure
Type of Microbial Population to be Destroyed
Environmental Conditions
o Temperature
o pH
o Type of Material on Which the Microorganism Exists
Chemical Agents of Control: Chemotherapeutic Agents
o Chemotherapeutic agents: chemical substances used in the treatment of infectious diseases
Purpose interfere with bacterial metabolism
Antibiotics synthesized and secreted by some true bacteria, actinomycetes, and fungi
that destroy or inhibit growth of other microorganisms
Synthetics drugs synthesized in the laboratory
o Kirby-Bauer Antibiotic Sensitivity Test
Purpose to determine the drug susceptibility of microorganisms isolated from infectious
processes

Rapid determination of efficacy of the drug by measuring the diameter of the zone
of inhibition that results from diffusion of the agent into the medium surrounding
the disc
Mueller-Hinton Agar (MH 2)
Softer agar to absorb ATB
Contains starch inhibits chemical released by microbes to prevent interference
with ATB
Measurement of the zone of inhibition in mm determines resistant, intermediate, or
susceptible to the ATB
Microbiology of Water
o Presumptive Test Detects coliforms by using a lactose fermentation broth + gas vial
Coliform
Rod
GRAM-NEGATIVE
Non-endospore forming
Ferments LACTOSE (RED YELLOW)
E.g. E.coli (GREEN METALLIC)
MPN an estimate of the number of coliforms present
o Confirmed Test Uses an EMB agar
EMB INHIBITS the growth of gram-POSITIVE bacteria

Lab 9 & 10 MORPHOLOGICAL STUDY OF UNKNOWN; DETERMINATION OF OXYGEN

REQUIREMENTS; CULTURAL CHARACTERISTICS; PHYSIOLOGICAL CHARACTERISTICS:
BIOOIXDATIONS, HYDROLYSIS, MISCELLANEOUS TESTS
Glucose
o Glucose
+
YELLOW
-
RED
o Gas
+
Lifted wax
-
Overlaying wax
Lysine
o +
VIOLET
o -
YELLOW
H2S/Indole
o Hydrogen
+
BLACK/BROWN
-
BEIGE
o Indole
+
PINK/RED
-
COLORLESS
Urea
o +
PURPLE
o -
BEIGE
Citrate carbon source for energy (enzyme citrate permease; citrase)
o +
BLUE
o -
GREEN

Lab 11-13 STAPHYLOCOCCI/STREPTOCOCCI: ISOLATION & IDENTIFICAITON

Staphylococcus
o Gram-positive
o Mesophile
o Non/pathogenic
o Non-spore forming
o Resistant to drying
o Examples
S. aureus
Skin infections
Mannitol Salt Agar ferments glucose (YELLOW)
Blood Agar beta-hemolytic (COMPLETE hemolysis, Clear zone)
Rabbit Plasma Coagulase Test Coagulase + (Cloudy)
S. epidermidis
Skin lesions; endocarditis
S. saprophyticus
UTI
MH2 Resistant (Novobiocin)
Streptococcus
o Gram-positive
o Round, chains
o Causes more diseases than any other group
o Require enriched media to survive
Examples
S. pyogenes
o Blood Agar beta-hemolytic
S. agalactiae
o Blood Agar beta-hemolytic
S. faecolis
o Blood Agar Alpha/gamma hemolytic
o 6.5% NaCl Broth (distinguishes enterococci from non-enterococci)
cloudy
o Esculin Bile Test + BLACK