BIO205: Lab Final
Which controls on the microscope affect the amount of light reaching the oculars?
Iris diaphragm
Visually identify a simple stain, negative stain, differential stain
Simple stain: crystal violet (remain same color) stains against light background
Negative stain: stains BACKGROUND
cells remain clear on dark background
Differential stains: different colors to distinguish different cells
State three things heat fixing a smear accomplishes
Kill bacteria
Adhesion (attach bacteria to microscope slide)
Preserve overall shape of cells
State the charge of simple stains and negative stains
Simple stains
Positively charged
Cells become stained
Crystal violet, safranin
Negative stains
Negatively charged
Stain background instead of cells
India ink, nigrosin
List, in order, the reagents used in the Gram stain procedure
CV: primary stains cells purple
Grams iodine: mordant (helps trap dye inside cells)
Alcohol: decolorizer (removes CV)
Safranin: counterstain pink/red
Gram neg turns pink
Gram positive remain purple
State the function of each reagent of each Gram stain reagent
Explain the mechanism of the gram stain (why do Gram negative cells lose the primary stain during decolorization- two reasons)
Thin peptidoglycan layer
Outer membrane is dissolved by decolorizer (alcohol)
Increases cell walls permeability
Allows CV-idione complex to wash out of cell
Correctly interpret and report the Gram reaction (Gram positive or Gram negative) and morphology (shape) of Gram-stained bacteria.
State the two genera that are identified using the acid-fast stain
Mycobacterium (mycolic acid in cell walls)
Nocardia
State the diseases caused by acid-fast bacteria
Tuberculosis
Leprosy
Nocardiosis
Distinguish acid-fast organisms from non-acid-fast organisms in a micrograph image
pink/red rods? = acid fast bacilli
Blue cells? = non acid fast
***** acid fast holds the red
***** non acid fast takes the counterstain
Explain why certain organisms are “acid-fast”
Gram stain: separates bacteria based on cell wall structure (peptidoglycan thickness)
Positive: PURPLE
Negative: PINK
Acid-fast stain: separates bacteria based on presence of mycolic acid
Acid fast: red/pink
Non acid-fast: blue
Lab 4: Handwashing
What types of microorganisms reside on our bodies? (vocabulary)
Microbiota
Why is there a concern for increased use of alcohol-based sanitizers?
Why did we “clamshell” the agar plates when performing this experiment?
Minimize contamination
Allow access to agar surface
Lab 5: Quantitation
Why are serial dilutions performed in microbiology lab experiments?
To obtain populations that are countable
Samples contain populations of organisms that are too large
Work backwards to determine population size of original sample
How many colonies must a plate have for it to be considered a countable plate?
30-300 colonies
Given a colony count, plate dilution, and inoculation amount, calculate the titer of cells in CFU/ml
Be able to do this with 1mL inoculation amount
Be able to do this with 0.1mL inoculation amount
Be able to write this answer in scientific notation
COUNT ZEROES then ADD zeroes to end of number
Lab 6: Eukaryotic Microorganisms Mold Growth on Bread and Fruit
You can get background information in your lecture materials as well, see eukaryotes and eukaryotic diseases
Mold breaks down and digests organic material
Are fungi prokaryotes or eukaryotes?
Eukaryotes
True nucleus
Membrane bound organelles
Chitin containing cells
Is mold unicellular or multicellular?
Unicellular yeast or multicellular mold
By what process does mold reproduce?
Spore formation
Asexual reproduction (air and grow into new mold)
Microscopic spores (similar to seeds by plants)
Lab 7: Disinfectants and Microbial Control
What is a disinfectant vs an antiseptic?
Disinfectant: kill or reduce microorganisms on nonliving surfaces
(disinfect door handle or dirty surfaces)
Antiseptic: kill or reduce microorganisms on living tissues SKIN
(anti on your anatomy)
Why can some chemicals be used as a disinfectant but not antiseptic?
Too harsh on living tissue or toxic
Disinfectants are stronger and can damage cells and harm humans
What is the difference between sanitization and disinfection?
Sanitization: SAFE - lowers germs to safe level
Does not always kill all microbes
Food, hands, dishes, everyday
Disinfection: DESTROY - kills more harmful microorganisms on surfaces
Lab surfaces, medical equipment, high touch areas
What are cellular features (things the cell has) that can help it resist disinfection?
Cell wall
Outer membrane
Cellular enzymes and repair mechanisms
Capsules
Endospores (resistant structures produced by some bacteria
Survive harsh conditions
Lab 8: Microbial Genetics DNA Extraction
Is DNA soluble in water?
yes
Is DNA soluble in alcohol?
No
Visually identify spooled DNA
Stringy, spooled, mucus like, cloudy
Lab 9: Aseptic Technique
What is aseptic transfer?
Moving microorganisms or sterile materials from one place to another while preventing contamination
What does it mean for a culture to be pure? Contaminated?
Pure culture: one type
Contamination: unwanted microorganisms in addition to the one being studied
Why is it important to work with sterile equipment when performing labs?
Contamination
Maintain pure culture
Environment safety
Accurate results
What does growth look like on an agar plate (refer to lab 5)? On a slant? In broth?
Agar plate = colonies
slant = streak of growth
Broth = turbidity, sediment
Lab 10: Streak Plates
You can get background information in your lecture materials as well, see microbial growth.
What is the purpose of a streak plate?
Isolate individual bacterial colonies and obtain pure culture
Spread cells out -> separate colonies -> pure culture
What does growth look like on an agar plate?
Colonies
Spots or clusters
Size
Color
Shape
Texture (smooth, rough, fuzzy)
Elevation (flat, raised, dome shaped)
Why are agar plates incubated upside down?
To prevent condensation from dripping onto agar surface
Describe how to perform a t-streak
-
What is the purpose of switching and using a new loop for each section?
Contamination
Reduce the number of bacterial cells being transferred
Separate individual cells
Prevent overcrowding of growth
NEW LOOP = FEWER CELLS = BETTER ISOLATION
Define: colony
Mound of cells genetically identical and free from other species or strains of bacteria
Lab 11: Biochemical Testing Experiment
Name the enzyme the oxidase test tests for
Cytochrome c oxidase
Do bacteria that produce ATP via aerobic energy production test positive or negative for oxidase? Explain.
Positive
Contains the complex cytochrome c oxidase
Part of the ETC during aerobic respiration
Do bacteria that produce ATP via anaerobic energy production test positive or negative for oxidase? Explain.
Negative
Anaerobic bacteria does not use oxygen as the final electron acceptor and does not usually use cytochrome c oxidase
Visually identify a positive oxidase test and understand the limits of reading the test results
Turn purple
What are the food sources for the TSI media? (sugars, proteins etc).
*** carbohydrates and proteins
Sugars
Glucose
Lactose
Sucrose
Protein
Peptones (broken down proteins/amino acids)
Other
Ferrous sulfate
Sodium thiosulfate
Phenol red
What are the products of fermentation?
Acids
Alcohols
Gases
Sugar -> fermentation -> acids + sometimes alcohols + gases
If given a TSI slant that has been incubated with E.coli interpret a slant that has turned yellow on the slant, butt, and cracked the media
What does black coloring mean in the TSI test?
Hydrogen sulfide production
Lab 12: Selective Media Experiment
You can get background information in your lecture materials as well, see microbial metabolism and microbial growth.
Explain how Mannitol salt agar is both a selective and differential media
Selective:
7.5% sodium chloride (salt)
High salt inhibits growth of most bacteria but allows salt tolerant bacteria to grow
Differential:
Sugar mannitol and pH indicator phenol red
Bacteria that ferments mannitol produce acid, lowering the pH and turns the medium yellow
Bacteria that does not ferment leaves the medium red/pink
*** selective media = “who can grow”
Contains something that blocks or slows down certain organisms while allowing others to grow
Look at growth v no growth
*** differential media = “what kind of organism is it?”
Allows multiple organisms to grow but causes them to look different based on biochemical reaction
Look at change in appearance
Visually identify positive and negative reactions on mannitol salt agar.
Positive
Color change = red/pink -> yellow
Growth occurs (bacteria can tolerate high salt)
Organism ferments mannitol, producing acid that lowers pH and turns the phenol red indicator to yellow
Negative
Growth may occur (organism is salt tolerant)
Agar remains pink NO COLOR CHANGE
Organism does not ferment mannitol, no acid produced
What pathogen appears with growth and yellow on MSA?
Staphylococcus aureus
Tolerates high salt concentration
What reagent was added to determine if bacteria produce catalase?
Hydrogen peroxide
Why are bubbles produced in a positive catalase test?
Enzyme catalase breaks down hydrogen peroxide into water and oxygen gas
Forms bubbles
Interpret a catalase test.
Bubbles present = catalase positive
Makes catalase enzyme
Able to break down hydrogen peroxide into water and oxygen
No bubbles = catalase negative
Does not produce catalase
Cannot break down hydrogen peroxide into oxygen
Explain why obligate aerobes do or do not produce catalase
Produce catalase
Require oxygen for growth and protect themselves from toxic oxygen byproducts
Obligate aerobes -> usually catalase positive bc they need enzymes like catalase to remove toxic oxygen compounds
Why is catalase useful to determine between Staphylococcus and Streptococcus?
Staphylococcus
Produce catalase +
Breaks down hydrogen peroxide into water and oxygen
Produce bubbles when added
Streptococcus
Do not produce catalase -
Does not break down hydrogen peroxide
Define: coliform (there are 3 characteristics that make an organism a “coliform)
Gram negative
Rod shaped bacilli
Ferment lactose with acid and gas production
Explain how MacConkey agar is both a selective and differential media
Selective:
Bile salts and crystal violet
Inhibit most gram positive bacteria
Allows gram negative bacteria to grow
Differential:
Sugar lactose and pH indicator neutral red
Ferment lactose produce acid = colonies pink/red
Do not ferment lactose = colorless or pale
SELECTIVE TELLS U WHO IS THERE. DIFFERENTIAL TELLS YOU WHAT THEY ARE DOING!
Visually identify positive and negative reactions on MacConkey agar
Positive = pink/red
Negative = colorless, clear, or pale
**** MacConkey = growth tells you it is likely gram negative, color tells you if it ferments lactose
Identify/name an organism that will grow metallic green on EMB
E. Coli = EMB green sheen
Strongly ferments lactose
Lab 13: Antibiotic Sensitivity
You can get background information in your lecture materials as well, see antimicrobials.
In Kirby Bauer antibiotic sensitivity testing, understand how zone size relates to antibiotic sensitivity
Large zone of inhibition: bacteria more susceptible (sensitive) to the antibiotic (EFFECTIVE)
Antibiotic prevents growth over a larger area
Small zone of inhibition: less sensitive or resistant to antibiotic
Bacteria can grow closer to antibiotic disk
No zone of inhibition: bacteria are RESISTANT to antibiotic (NOT EFFECTIVE)
Given an antibiotic sensitivity chart and a Kirby Bauer test, measure the zone of inhibition in mm and interpret the results.
Define and be able to identify: broad spectrum vs narrow spectrum antibiotics using a Kirby Bauer test. (we used E.coli and Staphylococcus if an antibiotic was effective on both, what does that tell you etc.)
Broad: antibiotic effective against wide variety of bacteria, often including both gram positive and gram negative bacteria
If an antibiotic creates ZOI around both E. Coli (gram neg) and Staph.(gram positive) = broad spectrum
Narrow: antibiotic effective against limited group of bacteria (specific types)
ZOI only one organism = narrow
Lab 1: Lab Safety
Personal Protective Equipment (PPE)
Required PPE: When performing lab exercises, you must wear a lab coat or apron, safety goggles or glasses, and disposable gloves. Closed-toe shoes and pulling back long hair are also standard requirements.
Lab Hygiene & Work Area Management
Disinfecting the Workspace: You must disinfect your lab work area with alcohol or 10% bleach before starting any lab work and after completing your exercises.
Glove Removal: Gloves should be removed immediately if they become contaminated, punctured, or torn. They must always be removed before leaving the laboratory space and discarded in designated biohazard waste bins.
Handwashing: Wash your hands thoroughly with soap and water before putting on gloves, immediately after removing gloves, and anytime you risk direct exposure to microbial cultures.
Labs 3a and b: Microscopy and Staining
Bright Field Microscopy
Parts and Functions:
Ocular Lens (Eyepiece): The lens you look through; typically magnifies the image 10\times.
Objective Lenses: Lenses on the rotating nosepiece (usually 4\times, 10\times, 40\times, and 100\times oil immersion) that provide the primary magnification.
Stage & Stage Clips: Holds the specimen slide securely in place.
Coarse Adjustment Knob: Moves the stage rapidly up and down for initial focusing (use only with low-power objectives).
Fine Adjustment Knob: Permits precise, slight stage movements for sharp focusing under high power.
Condenser: Focuses and concentrates light from the illuminator onto the specimen.
Iris Diaphragm: Regulates the amount of light passing through the condenser to adjust contrast and resolution.
Calculating Total Magnification: \text{Total Magnification} = \text{Magnification of Ocular Lens} \times \text{Magnification of Objective Lens}
Light Regulation Controls: The iris diaphragm, condenser height, and the light intensity dial (rheostat) directly affect the amount of light reaching the oculars.
Staining Principles & Smear Preparation
Visual Identification:
Simple Stain: Uses a single basic dye; all cells appear the same color against a bright background.
Negative Stain: Cells remain clear/uncolored while the background is darkened by an acidic dye.
Differential Stain: Uses multiple dyes to distinguish between different types of bacteria or structures (e.g., Gram stain, Acid-fast stain).
Three Purposes of Heat Fixing:
Kills the microorganisms.
Adheres (fixes) the cells firmly to the glass slide so they don't wash off.
Coagulates cytoplasmic proteins to make them more receptive to stains.
Stain Charges: * Simple stains (Basic dyes): Positively charged (\text{cationic}) chromophores that bind to the negatively charged bacterial cell wall.
Negative stains (Acidic dyes): Negatively charged (\text{anionic}) chromophores that are repelled by the cell wall, staining only the background.
The Gram Stain Procedure
Primary Stain (Crystal Violet): Stains all bacterial cell walls purple.
Mordant (Gram's Iodine): Forms a large water-insoluble crystal violet-iodine (\text{CV-I}) complex inside the cell.
Decolorizer (95% Ethanol/Acetone): Washes out the dye complex from Gram-negative cells; Gram-positive cells retain it.
Counterstain (Safranin): Stains the decolorized Gram-negative cells pink/red.
Mechanism of Decolorization (Why Gram-Negatives Lose the Primary Stain):
Gram-negative cell walls have a high lipid content in their outer membrane. Ethanol dissolves this lipid layer, making the wall porous.
Gram-negative cells have a very thin layer of peptidoglycan, which cannot trap the large \text{CV-I} complexes once the outer membrane is breached.
Interpretation: * Gram-positive: Purple cells.
Gram-negative: Pink/red cells.
Morphologies: Cocci (spheres) or Bacilli (rods).
Acid-Fast Stain
Genera Identified: Mycobacterium and Nocardia.
Diseases Caused: Tuberculosis (Mycobacterium tuberculosis) and Leprosy (Mycobacterium leprae).
Visual Distinction: Acid-fast organisms appear bright pink/red (due to carbolfuchsin retention), while non-acid-fast organisms stain blue (methylene blue counterstain).
Why they are "Acid-Fast": These bacteria possess a thick, waxy outer cell wall rich in mycolic acids, which resists conventional water-based stains but holds tightly to lipid-soluble dyes even when washed with acid-alcohol.
Lab 4: Handwashing
Microorganisms on the Body: Resident microflora (normal flora that permanently colonize deep skin layers) and transient microflora (microbes picked up temporarily from environment contact).
Alcohol-Based Sanitizer Concerns: Over-reliance can strip protective skin lipids leading to irritation, fails to eliminate certain resilient non-enveloped viruses or bacterial endospores (like C. difficile), and can drive selective pressure toward sanitizer-tolerant strains.
"Clamshell" Technique: Opening the petri dish lid at a slight angle rather than removing it completely minimizes the exposure of the sterile agar surface to airborne contaminants.
Lab 5: Quantitation (Serial Dilutions)
Purpose of Serial Dilutions: To reduce highly dense bacterial concentrations step-by-step down to an countable range, enabling the isolation of distinct, single colonies.
Countable Plate Range: A plate must have between 30 and 300 colonies to be statistically valid for calculation.
Calculating Titer (\text{CFU/mL}): \text{Titer (CFU/mL)} = \frac{\text{Number of Colonies (CFU)}}{\text{Volume Inoculated (mL)} \times \text{Plate Dilution Factor}}
Practice Examples
Example 1: 1 mL Inoculation
Data: 150 colonies, Plate Dilution = 10^{-5}, Inoculation Volume = 1.0\text{ mL}.
Calculation: \frac{150}{1.0 \times 10^{-5}} = 150 \times 10^{5}
Scientific Notation: 1.5 \times 10^{7}\text{ CFU/mL}
Example 2: 0.1 mL Inoculation
Data: 75 colonies, Plate Dilution = 10^{-6}, Inoculation Volume = 0.1\text{ mL}.
Calculation: \frac{75}{0.1 \times 10^{-6}} = \frac{75}{10^{-7}} = 75 \times 10^{7}
Scientific Notation: 7.5 \times 10^{8}\text{ CFU/mL}
Lab 6: Eukaryotic Microorganisms (Mold Growth)
Fungi Classification: Fungi are eukaryotes (they contain a true nucleus and membrane-bound organelles).
Mold Structure: Mold is a multicellular fungus comprised of filaments called hyphae.
Reproduction: Molds reproduce primarily by forming asexual or sexual spores (such as conidiospores or sporangiospores) that disperse via wind or water.
Lab 7: Disinfectants and Microbial Control
Disinfectant vs. Antiseptic:
Disinfectant: A harsh chemical agent applied to non-living surfaces (fomites) to destroy or inhibit vegetative microbes.
Antiseptic: A milder chemical agent formulated safe enough to be applied to living tissues (skin, wounds).
Why Chemical Uses Differ: Certain strong chemicals destroy cellular membranes or denature proteins indiscriminately; they are highly effective on steel countertops but toxic or corrosive to human tissue.
Sanitization vs. Disinfection: * Sanitization lowers microbial counts to safe, public-health regulated levels through mechanical cleaning.
Disinfection actively kills vegetative pathogenetic microflora on a surface, though it may spare endospores.
Resistance Features: Structural components such as bacterial endospores, thick waxy mycolic acid layers (acid-fast walls), outer membranes of Gram-negative bacteria, and biofilms increase resistance to chemical disinfectants.
Lab 8: Microbial Genetics (DNA Extraction)
Water Solubility: DNA is soluble in water due to its highly polar, negatively charged phosphate backbone.
Alcohol Solubility: DNA is insoluble in alcohol (like ice-cold ethanol or isopropanol). Alcohol dehydrates and precipitates the DNA out of solution.
Visual Identification: Spooled DNA appears as clear, viscous, stringy/web-like threads wrapped around a glass rod or plastic loop.
Lab 9: Aseptic Technique
Aseptic Transfer: The sterile, controlled movement of a microbial culture from one vessel to another without contaminating the environment, the handler, or the source culture.
Culture Definitions:
Pure Culture: A container holding only a single, isolated species of microorganism.
Contaminated Culture: A culture that has accidentally acquired unwanted foreign microorganisms.
Importance of Sterility: Prevents false experimental outcomes, protects lab personnel from exposure, and maintains pure cultures for downstream testing.
Visual Growth Profiles:
Agar Plate: Well-defined, isolated circular or irregular mounds (colonies).
Agar Slant: A visible film or streak of growth along the angled surface (can be film-like, spreading, or beaded).
Broth: Indicated by turbidity (cloudiness), pellicle formation (surface film), or sediment (cells pooled at the bottom).
Lab 10: Streak Plates
Purpose: To mechanically dilute a bacterial sample across an agar plate to obtain isolated, individual colonies from a mixed culture.
Inversion of Agar Plates: Plates are incubated upside down to prevent condensation from forming on the lid and dripping down onto the agar, which would cause colonies to run together and ruin isolation.
Performing a T-Streak: Divide the plate into three distinct regions forming a "T". Streak your initial sample heavily into Zone 1. Flame your loop, cool it, drag it through Zone 1 twice, then zig-zag through Zone 2. Flame and cool again, drag through Zone 2 twice, and zig-zag into Zone 3.
Purpose of a New Loop/Flaming: It kills remaining bacteria on the loop between steps, ensuring you are strictly diluting the cells deposited from the previous section rather than adding more.
Colony Definition: A visible cluster of millions of identical cells originating from a single mother cell (Colony Forming Unit).
Lab 11: Biochemical Testing Experiment
Oxidase Test
Enzyme Tested: Cytochrome c oxidase.
Aerobic Energy Producers: Test positive. These bacteria use oxygen as their terminal electron acceptor in the electron transport chain, requiring cytochrome c oxidase.
Anaerobic Energy Producers (Strict Anaerobes/Fermenters): Test negative. They utilize alternative terminal electron acceptors or pathways that skip cytochrome c oxidase entirely.
Visuals & Limits: A positive test turns deep purple/blue within 20 seconds. If left too long, ambient oxygen in the air can auto-oxidize the reagent, causing a false positive.
Triple Sugar Iron (TSI) Agar
Food Sources: Contains three sugars (Glucose, Lactose, and Sucrose) and proteins (peptones/beef extract).
Products of Fermentation: Organic acids (which lower pH) and gases like \text{CO}_2 and \text{H}_2.
E. coli Reaction Interpretation: A yellow slant, yellow butt, and cracked media indicates glucose, lactose, and/or sucrose fermentation (acid/acid) along with gas production (cracks/lifts in media).
Black Coloring: Indicates Hydrogen Sulfide (\text{H}_2\text{S}) production. The gas reacts with iron components in the media to form a black precipitate (ferrous sulfide).
Lab 12: Selective Media Experiment
Mannitol Salt Agar (MSA)
Dual Function:
Selective: High salt concentration (7.5\%\text{ NaCl}) inhibits most bacteria except halotolerant species like Staphylococcus.
Differential: Contains the sugar mannitol and phenol red pH indicator. Organisms that ferment mannitol produce acid, changing the agar color from red to yellow.
Visual Interpretation: Growth with yellow halo = Positive mannitol fermenter; Growth with remaining red/pink agar = Non-mannitol fermenter.
Key Pathogen: Staphylococcus aureus grows well and turns the medium yellow.
Catalase Test
Reagent Added: Hydrogen peroxide (\text{H}_2\text{O}_2).
Why Bubbles Form: Catalase breaks down toxic hydrogen peroxide into water and oxygen gas (\text{O}_2), creating instant bubbling. 2\text{H}_2\text{O}_2 \xrightarrow{\text{Catalase}} 2\text{H}_2\text{O} + \text{O}_2 \uparrow
Aerobe Production: Obligate aerobes do produce catalase because oxygen metabolism naturally generates toxic superoxide and hydrogen peroxide byproducts that the cell must detoxify to survive.
Diagnostic Use: Differentiates Catalase-positive Staphylococcus from Catalase-negative Streptococcus.
MacConkey Agar (MAC) & EMB Agar
Coliform Definition: Gram-negative, rod-shaped bacteria that ferment lactose with the production of acid and gas.
MacConkey Dual Function:
Selective: Bile salts and crystal violet inhibit Gram-positive bacteria, selecting for Gram-negatives.
Differential: Lactose and neutral red indicate fermentation. Lactose fermenters form pink/red colonies, while non-fermenters form colorless/white colonies.
EMB Metallic Green Growth: Characteristic of Escherichia coli (due to rapid, vigorous lactose fermentation yielding heavy acid accumulation).
Lab 13: Antibiotic Sensitivity
Kirby-Bauer Method
Zone Size Relationship: The larger the Zone of Inhibition (the clear area around an antibiotic disc where bacteria cannot grow), the more sensitive the bacteria are to that antibiotic. Small or absent zones indicate bacterial resistance.
Measuring and Interpretation: Measure the diameter of the clear zone in millimeters (\text{mm}). To interpret, compare your measurement against a standardized reference chart to classify the organism as Resistant (R), Intermediate (I), or Susceptible (S).
Broad vs. Narrow Spectrum:
Broad-spectrum: An antibiotic that kills or inhibits a wide variety of both Gram-positive and Gram-negative bacteria (e.g., effective against both E. coli and Staphylococcus).
Narrow-spectrum: An antibiotic that targets only a specific subgroup of bacteria (e.g., effective only against Gram-positives like Staphylococcus but ineffective against E. coli).