Microbiology Lab Midterm Study Guide

Review all chapters in your exercise manual beginning with the introduction
INTRODUCTION: (Covered 9.4.14 II Week 1)
Biosafety levels1: basic level of containment. Hand washing or wearing gloves
2: Appropriate for working with human body fluids. Autoclave, sharps containers, lab coats
3: appropriate for working with pathogens that can be transmitted via respiratory route. Self-closing, double doors and sealed windows
4: Highest level. Aerosol pathogens; pathogens with no vaccine/treatment. Separate building with separate ventilation and waste management systems
Bacteria used in lab is classified as Class 1 by US Public Health Service/Biosafety Level 2
Review everything about the microscope and our original laboratory
EXERCISE 1: USE AND CARE OF MICROSCOPE: (Covered 9.4.14 II Week 1)
THE MICROSCOPE:
Simple microscope early microscope; like a magnifying glass
Brightfield compound microscope Most often used
Base Bottom of microscope
Stage Holds slide
Arm Carries the microscope
Body Tube Transmits the magnified image
Condenser Lenses that concentrate light by focusing it into a cone
Iris Diaphragm Controls the angle and size of light cone
Objective lenses Magnify specimen
Ocular/eyepiece lens: 10x
Monocular One lens
Binocular Two lens
FOCUSING THE MICROSCOPE:
Coarse adjustment Focuses low power objectives (4x and 10x)
Fine adjustment Focusing with high-power (40x and 100x)
Field of vision Area seen through microscope
Magnification Depends on lens being used
Oil immersion lens 100x
RESOLUTION:
Resolving power/resolution Ability of lenses to reveal fine detail or two points distinctly separated
Numerical aperture The lens system
Refractive index The amount the light bends
Focal point Where an image is formed
Spherical aberration Fuzzy periphery due to curvature of lens, resulting in multiple focal points
Chromatic aberration Multitude of colors due to different wavelengths of light passing through lens
Monochromatic light Lightsource with one wavelength of light eliminates aberrations
Parfocal When a subject is in focus with all lenses

3) Understand the hanging drop and temporary wet mount and slides presented for observation
EXERCISE 2: EXAMINATION OF LIVING MICROORGANISMS: (covered 9.11.14 II Week 2)
Wet Mount and Hanging Drop were performed
PERFORM A WET MOUNT
Use pipette to transfer drop of sample to a slide
Cover sample with cover slip
PERFORM A HANGING DROP
Cover edges of coverslip with petroleum jelly
Place cover slip face-up on white paper towel
Transfer small drop of sample on coverslip
Plate depression slide over drop
VOCABULARY:
Brightfield microscopy Objects are dark and the field is bright
Phase-contrast microscopy A ring of light is passed through the object and light rays are diffracted
Diffracted Pushed aside
Brownian movement Not true motility but movement caused by molecules in liquid striking an object & causing it to shake or bounce
What type of organisms could we expect to see in this lab?
Mainly protozoans. Some examples:
ALGAE PROTOZOA
Euglena
Diatoms
Chlamydomonas
Spirogyra
Volvox
Scenedesmus Paramescium
Amoeba
Stylonychia
Heteronema
Vorticella
Which type of compound microscope do we use in lab?
EXERCISE 3: MICROBES IN THE ENVIRONMENT (9.18.14 II Week 3)
BACTERIAL MORPHOLOGY: (Page 5 of Supplemental Guide)
MORPHOLOGICAL DESCRIPTION ASSOCIATED GENUS
Cocci that divide in one plane, forming a chain of cocci Streptococcus
Cocci that divide in three planes at right angles, forming cubical packets Sarcina
Cocci that divide in two planes, forming tetrads Gaffyka
Cocci that divide in two planes, forming irregular clusters 1. Staphyloccus 2. Micrococcus
DESCRIPTION BACTERIAL FORM
Bacilli with tapered ends, like a cigar Fusiform bacilli
Bacilli that resemble cocci Coccobacilli
Curved rods resembling commas, form S-shaped strands Vibrios
Spiral-like corkscrews; rigid spirilla
Flexible spirals, with contractile axial filaments Spirochetes
As bacteria age, they assume odd shapes, such as swelling or rudimentary branching, difficult to determine shape of bacteria Involution forms
VOCABULARY:
Chemically defined media A medium whose exact chemical composition is known
Complex media Media for which the chemical composition varies slightly from batch to batch
Nutrient broth Liquid complex medium
Nutrient agar Solid medium
Agar An extract from marine red algae
Steam sterilization/autoclaving Most common method of sterilizing media
Petri plates Provide a large surface area for examinating colonies
Inoculated Intentionally inoculate
Incubation period Period of time in which bacteria increase
Turbid Cloudy, representing bacterial growth
Colony Population of cells that arises from a single bacterial scell
Colony-forming unit A group of the same microbes attached to one another
Sediment Microbial cells settled at the bottom of the tube
Flocculent Clumps of microbial cells
Pellicle Membrane at surface of broth
COLONY DESCRIPTIONS
WHOLE-COLONY APPEARANCE
Circular
Irregular
Biconcave (embodied in agar)
Filamentous
Rhizoid (long projections)
MARGIN (EDGE)
Entire
Undulate
Lobate
Filamentous
Curled
MARGIN (EDGE)
Flat
Raised
Convex
Umbonate

What type of bacteria did we use in this lab?
# 6: Streptococcus fecalis in sterile nutrient broth
What was seen? Turbidity
# 7: Pseudomonas aeruginosa in nutrient slant
What was seen? Bluish-green film on NA slant
# 8: Proteus vulgaris in sterile nutrient slant
What was seen? **Pellicle
** Need to check on “what was seen” with other classmates to confirm
Understand transfers, media, all techniques used in the laboratory
EXERCISE 4 TRANSFER OF BACTERIA: ASEPTIC TECHNIQUE (9.18.14 II Week 3)
PERFORM ASEPTIC TECHQNIUE
Sterilize loop in blue flame
While holding loop and bacterial culture, remove cap with pink of hand holding loop
Briefly pass mouth of tube through flame three times before inserting loop
Get a loopful of culture
Heat the mouth of the tube again and replace cap
VOCABULARY
Contaminants Unwanted microbes
Aseptic technique Technique used to exclude contaminants
Sterilized Rendered free of all life
Broth cultures Large numbers of bacteria in a small space
Agar slants Solid culture media at an angle
Agar deep Solidified agar in a test tube
Inoculating loop Loop used to transfer bacteria during aseptic technique
Inoculating needle Needle used during aseptic technique
PATTERNS OF GROWTH ON AGAR SLANTS
Arborescent (branched)
Beaded
Echinulate (pointed)
Filiform (even)
Rhizoid (rootlike)
Spreading (effuse)

5) Simple stains: technique, reagents, organisms used and results
EXERCISE 5: PREPARATION OF SMEARS AND SIMPLE STAINS (9.25.14 II Week 4)
PERFORM A SIMPLE STAIN:
Heat fix bacterial slide
Apply methylene blue for 1 min
Rinse
Blot dry
Observe slide under microscope
VOCABULARY:
Acid fast Used to distinguish Mycobacterium and some Nocardia species. Acid fast=red Non-acid fast=blue
Negative stain For finding capsules. Unstained halos=capsules
Endospore Malachite green turn endospores green; remainder of cells are pink or red
Flagella Mordant used to build up diameters of flagella until they’re seen and then stained by carbolfuchsin
Smear A thin film of bacterial cells
Heat-fix Passing dry smear through Bunsen burner several times
Chemically-fix Cover slide with 95% methanol solution for 1min
Chromophore The ion that is colored
Basic stain If chromophore is positive like Methylene blue
Acidic stain If it’s negative ion
Simple stain Staining procedure using only one stain; usually basic dye
Direct stain Simple stain that stains bacteria
Negative stain Simple stain that stains background
What’s the purpose of a simple stain?
Determine morphology, size, and arrangement
Which bacteria were used during the Simple Stain lab and what were the results?
BACTERIA WHAT I SAW:
1: Staphylococcus epidermis Blue cocci
2: Bacillus subtilis Blue rods
3: Esherichiae coli Blue cocci
14: Bacillus cereus Blue rods
Gums Blue cocci
Capsule stain: technique, reagents, organisms used and results
EXERCISE 6: NEGAVITVE STAINING (10.16.14 II Week 6)
PERFORM A NEGATIVE STAIN
Add India Ink on slide
Spread with spreader slide
Let air dry
Observe under microscope
Which bacteria were used during the Negative Stain lab and what were the results?
BACTERIA WHAT I SAW:
Carrot slime Halo effect/capsule
5: Enterobacter aeorgenes capsule
12:Staphyloccocus aureus capsule
**check with classmates regarding results
What is the purpose of a negative slide?
Used to demonstrate presence of capsules
Gram stains: technique, reagents, organisms used and results
EXERCISE 7: GRAM STAINING (9.25.14 II Week 4)
PERFORM A GRAM STAIN
HFBS
Apply Crystal Violet (30-60s)
Apply iodine (10-30s) to form CVI complex
Rinse
Apply Gram Decolorizer: Acetone alcohol. 10 drops at a slant
Rinse IMMEDIATELY!
Apply Safranin counterstain 30-60 sec
Rinse
Blot dry
VOCABULARY
Gram stain Differential stain
Primary stain Crystal violet. Dyes all bacteria purple
Mordant Gram’s iodine. Combines with Crystal violet to form CV-I (crystal violet iodine complex)
Decolorizing agent Ethanol. Primay stain is washed out of some bacteria
Secondary stain/counterstain Safranin. Stains decolorized bacteria red
Gram-negative Decolorized easily
Gram-positive Decolorized slowly
Peptidoglycans Complex lattice structure that composed bacterial cell wall
Which bacteria were used during the Gram Stain lab and what were the results?
BACTERIA WHAT I SAW:
1: Staphylococcus epidermis Gram + cocci
2: Bacillus subtilis Gram + rods
3: Esherichiae coli Gram – rods
14: Bacillus cereus Gram + rods
Gums Gram + and - rods
Acid-Fast stain: technique, reagents, organisms used and results
EXERCISE 8: ACID-FAST STAINING (10.2.14 II Week 5)
PERFORM AN ACID-FAST STAIN:
Smear is flooded with carbolfuschin, which has high affinity for mycolic acid
Steam bath for 5 mins
Washed with acid-alcohol mixture to decolorize non-acid fast
Methylene blue counterstain for non-acid fast
VOCABULARY
Mycolic acid Wax-like lipid found in acid-fast organisms
Ziehl-Neelsen procedure Acid-fast procedure using steam to heat fix acid-fast bacteria
Kinyoun modification “cold stain” concentration of phenol in carbolfuschin is increased so heating is not necessary
Which bacteria were used during the Acid-Fast Stain lab and what were the results?
BACTERIA WHAT I SAW:
3: Escherichiae coli Non acid-fast. Blue bacilli
4: Mycobacterium smegmatis Acid-fast. Red/pink cocci
8) Spore stain: technique, reagents, organisms used and results
EXERCISE 9 + handout: STRUCTURAL STAINS (endospore, capsule, and flagella) (10.16.14 II Week 6)
PERFORM A SPORE STAIN
Add malachite green to slide-1 minute
Cover with a paper towel
Steam for 7 minutes continuously
Remove paper and wash with water (no decolorizer)
Counterstain with Safranin for 1 minute
Wash with water & blot dry
VOCABULARY
Structural stains Can be used to ID and study bacteria
Endospores Formed by several genera in the orders Bacillales and Clostridiales
Schaeffer-fulton The spore stain we did in class
Capsule A viscous coat round/oval in shape
Slime layer Irregularly shaped and loosely bound viscous coat around bacteria
Motile Ability to move from one position to another
Virulence Disease-causing ability
Flagella Most common means of mobility
Peritrichous Flagella all over
Monotrichous One flagellum at one end
Lophotrichous Lots of flagella at one end
Amphitrichous One flagellum at each end
Polar At one end or both ends of the cell
Which bacteria were used during the Spore Stain lab and what were the results?
BACTERIA WHAT I SAW:
2: Bacillus subtlis Pink rods and greenish/blue spores
14: Bacillus cereus Pink rods and greenish/blue spores
3: Escherichiae coli All vegetative/pink cocci
EXERCISE 11: ISOLATION OF BACTERIA BY DILUTION TECHNIQUES (9.18.14 II Week 3) & (9.25.14 II Week 4)
POUR PLATE CONCEPT:
Initial sample of 1 + 8 mixture (Staphylococcus epidermis and Proteus vulgaris) is diluted through a series of transfers
Final dilution is mixed with warm agar in Petri dish
Individual colonies are formed in an on the agar
STREAK PLATE CONCEPT:
1 + 8 mixture
Streaking from quadrant A to quadrant B, then from quadrant B to quadrant C, and then quadrant C to D.
Isolation of colonies should be seen after incubation
FOUR DIFFERENT TYPES OF STREAKING:
* Two types of quadrant streaking: the way we did it in lab and starting from quadrant B and going down
* Radiant Streaking: looks like grill marks
*Continuous Streaking: starting at quadrant A and just going back and forth all the way through the dish
EXERCISE 33: FUNGI: YEASTS AND MOLDS (covered 9.11.14 II Week 2)
Wet Mount and Hanging Drop performed
YEASTS AND MOLDS
Nucleus Membrane bound organelle housing DNA in eukaryotes
Organelles Membrane-bound, specialized structures and functions
Fungi Group that possess eukaryotic cells
Yeasts Nonfilamentous, unicellular fungi; normally spherical or oval
Psuedohypha When bud fails to detach; forms a short chain of cells
Molds Multicellular filamentous fungi
Thallus Microscopic mold colony
Mycelia Mass of strands that makes up thallus
Hypha Each strand that makes up mycelia
Vegetative hypha Growth on the surface of growth medium
Aerial/Reproductive hypha Originate from vegetative hyphae and produce asexual spores
Spores Aids in asexual reproduction
Septum Cross wall that separates hyphal strand
Septate hyphae Hyphae with septum
Ceonocytic hyphae Hyphae with no septum
IDENTIFICATION
Oomycota Fungus-like algae; growth requirements like fungi; aquatic environment
Zoospores Asexual spores of oomycota
Zygomycota Saprophytic molds that have coencytic hyphae
Saprophyte Contains nutrients from dead organic matter
Sporangium Asexual spores of Zygomycota; spore sac
Sporangiospores Spores inside of sporgangium
Zygospores Fusion of two cells. Sexual reproduction of Zygomycota
Ascomycota Mold with septate hyphae and some yeasts
Ascospores sexual spores of Ascomycota
Ascus Sac holding sexual spores
Conidiospores Asexual product of Ascomycota
Basidiomycota Fleshy fungi or mushrooms
Basidiospores Sexual spores basidium club-shaped stalk that produces sexual spores
Pathogens Disease-causing in plants and animals
Sabouraud agar Selective medium used to culture fungi
FUNGI
PHYLUM FUNGI ASEXUAL SEXUAL EXAMPLE
Zygomycota (conjugation fungi) Coenocytic hyphe Sporangiospores Zygospores Rhizopus (bread mold)
Ascomycota (sac fungi) Septate hyphae; yeastlike Conidiospores, budding Ascospores Aspergillus and Penicillium
Basidiomycota (club fungi) Septate hyphae; includes fleshy fungi (mushrooms) Fragmentation basidiospores FUNGUS-LIKE ALGAE
PHYLUM FUNGI ASEXUAL SEXUAL EXAMPLE
Oomycota (water molds) Coenocytic hyphae; cellulose cell walls Zoospores oospores Phytophthora infestans (Potato famine)
EXERCISE 34: PHOTOTROPS: ALGAE AND CYANOBACTERIA (covered 9.11.14 II Week 2)
Wet Mount and Hanging Drop performed
Algae Photsyntehtic eukaryote with no true roots, stems, or leaves; mainly in or near water
Cyanobacteria Bacteria that are often observed in water samples along with algae
CHARACTERISTICS OF MAJOR GROUPS OF PHOTOROPHS FOUND IN FRESHWATER
BACTERIA ALGAE
Characteristic Chyanobacteria Euglenoids Diatoms Green Algae
Chlorophyta Oomycotes
Cell Wall Blue-green Green Yellow-brown Green White
Cell type Bacteria-like Lacking Visible with regular-markings Visible Visible
Flagella Absent Present Absent Present with some On zoospores
Cell Arrangement Unicellular or filamentous Unicellular Unicellular or colonial Unicellular, colonial, or filamentous Filamentous
Nutrition Autotrophic Facultatively heterotrophic Autotrophic Autotrophic Heterotrophic
Produce Oxygen Yes Yes Yes Yes No
Examples Anabaena
Oscillatora Euglena Pinnularia, Diatoma, Cyclotella, Naviula Spirogyra, chlamydomonas, chlosterium Phytophthora infestans (Potato famine)
EXERCISE 35: PROTOZOA (covered 9.11.14 II Week 2)
Wet Mount and Hanging Drop Performed
VOCABULARY:
Protozoa Heterotrophic unicellular eukaryotic organism
Pellicle Outter covering, elastic membrane covering protozoa
Contractile vacuole Fills with freshwater and then contracts to eliminate excess water
Amebas Type of protozoa that moves via psueodopods
Pseudopods Cytoplasm of amoeba which helps in its mobility
Euglenozoa Have one more more flagella
Ciliates Cilia extend from cell
Ciliphora Phylum of ciliates
Oral groove Where food is taken up via cytostome
Food vacuole Forms in cytopharynx
Diplomonads Have multiple flagella and lack mitochondria; two nuclie; live in digestive tracts
Parabasalids Symbionts in many animals; Have multiple flagella and lack mitochondria apicomplexa Nonmotile obiligate intracellular parasitesCOMMON TYPES OF PROTOZOANS:
1. amoeba
2. Euglena
3. Paramecium