Microbiology Lab Exam 1

Effective hand washing is important for minimizing direct person-to-person and indirect contact transmission of pathogens. It is critical for laboratory professionals to minimize transmission to ______, ________, and contamination of __________.

others, inoculation of oneself, and contamination of cultures

In which other professions would reducing the transmission of potential pathogens be considered important?

• healthcare

• food handlers

• veterinary clinics

In the experimental results provided in this lab, which areas of the hands were typically the most difficult to clean, based on the individual’s hand-washing technique?

• area under the fingernails

• back of hands

• between fingers

Lab 1-1 GLO Germ Hand Washing Exercise Overview

Goal: training aid for people to learn how to wash their hands more effectively

Steps: hands are covered with lotion (contains minute plastic particles), rub hands together to spread lotion, use UV to see spread, wash hands, use UV again to see remaining germs present

What is the single most effective way to prevent the spread of germs?

handwashing

How does the refractive index of immersion oil compare with that of glass?

Immersion oil has the exact same refractive index as glass

• they have the same optical density meaning that the speed of light through immersion oil is equivalent to glass

• this prevents the loss of light due to refraction

How do you calculate the total magnification of a specimen seen through a compound light microscope? Give an example using the 4X objective.

Total magnification = magnification of objective lens x magnification of ocular lens

ex: 4x objective lens → 4 × 10 = 40x total magnification

Differentiate between the following terms: parfocal, binocular, and compound microscope

Parfocal = the microscope’s ability to stay in focus when switching between objective lenses. requires only minor adjustments (fine focus knob and illumination).

Binocular = a microscope with 2 eye pieces; provides 3D vision.

Compound Microscope = uses more than 1 lens to magnify an object. has objective lenses & ocular lens to achieve higher magnification.

Differentiate between the terms magnification and resolution

Magnification = involves enlarging an object’s appearance.

Resolution = clarity of an image; improves with smaller limit of resolution.

Limit of resolution (resolving power) = measurement of how far apart 2 points must be for the microscope to view them as separate. determined by wavelength/numerical aperture of condenser & objective lens. the smaller the wavelength, the lower the limit of resolution and the higher the actual resolution.

Why is it advisable to start with the low power lens first when viewing a slide through the microscope?

To allow you to explore the slide and find + center the object that you are planning to study

• also to ensure that the specimen is actually present

If you want to observe bacteria on a stained smear prep slide, at what point during the procedures would you add immersion oil to a microscope slide?

After focusing and centering the specimen under the 40x objective, but before switching to the 100x objective (oil immersion lens).

Lab 3-1 Introduction to the Light Microscope Overview

Purpose: To become familiar with the operations and limitations of a light microscope. Also, to examine two practice slides and learn about microscope functionality.

Steps: properly transport & clean microscope, turn on, start in 4x, move stage with mechanical slide holder, adjust diaphragm for light, coarse-focus knob, then 10x, coarse-focus, 40x, fine-focus, oil, 100x

• when done, make sure to lower the stage and remove the slide

Refraction

The bending of light as it passes through the objective lens from the specimens that produces a magnified real image

What is the best limit of resolution achieved by a light microscope?

About 0.2 micrometers

Field of view

the visible area when looking through the microscope eyepiece at the specimen

Dark-field microscopy

special condenser used so light reflected off the specimen only enters the objective lens

• brightly lit specimen against dark background

Phase contrast microscopy

special optical components to exploit subtle differences in the refractive indices of water & cytoplasmic components to produce contrast

• light waves

Fluorescence Microscopy

fluorescent dye that emits fluorescence when illuminated with UV.

Ocular lenses

to remagnify (10x) the image produced by the objective lens

Revolving nosepiece

holds the objective lenses & allows for rotation to change power

Objective lenses

to magnify the specimen and produce a real image

• 4x = scanning power

• 10x = low power

• 40x = high dry power

• 100x = oil immersion power

Arm

attaches body of the microscope to its base

Stage Clip

holds the microscope slide in place on the stage and prevents it from moving while the specimen is being viewed

Coarse-Focus Knob

moves the stage up and down the focus the image

• brings specimen into general focus

Fine-Focus Knob

slightly moves the stage up and down for focusing

• handles small & precise movement of the specimen

Mechanical Stage Adjustment Knob

controls the movement of the stage and allows you to position the slide within the microscope’s field of view

Stage

flat platform where the microscope slide is placed for viewing

Lamp

light source so that the specimen can be viewed

Iris Diaphragm

controls the amount of light entering the condenser

Condenser Lens

concentrates the light and makes illumination of the specimen more uniform

What are the three domains? Identify whether each domain contains prokaryotes or eukaryotes.

• Bacteria = prokaryotes

• Archaea = prokaryotes

• Eukarya = eukaryotes

What is the domain, classification group (protozoa, algae, or fungi), and mode of nutrition (heterotrophic or autotrophic) for paramecium?

domain = unicellular eukarya

classification group = protozoa (ciliates)

mode of nutrition = heterotrophic → searches for food in marine/freshwater

What is the domain, classification group (protozoa, algae, or fungi), and mode of nutrition (heterotrophic or autotrophic) for euglena?

domain = eukarya

classification group = algae

mode of nutrition = photosynthetic autotroph when light is available but heterotroph when light is not available. Known as a mixotroph.

What is the domain, classification group (protozoa, algae, or fungi), and mode of nutrition (heterotrophic or autotrophic) for spirogyra?

domain = eukarya

classification group = filamentous algae

mode of nutrition = photosynthetic autotroph

What is the domain, classification group (protozoa, algae, or fungi), and mode of nutrition (heterotrophic or autotrophic) for stentor?

domain = eukarya

classification group = protozoa (cilitates)

mode of nutrition = heterotrophic → in freshwater, can swim, but normally are anchored to a surface feeding

• larger than paramecium

Identify the domain and kingdom for molds and yeasts. What are the differences between molds and yeast?

domain = nonmotile eukarya

kingdom = fungi

molds = multicellular; grow as hyphae

yeasts = unicellular; reproduce by budding

Differentiate between the terms hyphae and mycelia as they relate to fungi.

hyphae = the individual, thread-like filaments of cells that mold grow as

mycelia = interwoven masses of hyphae; the vegetative portion of a fungi

What are the four supergroups that eukaryotes are divided into?

• Unikonta = closer to root of phylogenic tree; composed of heterotrophs & includes amoebas + fungi

• Excavata = unicellular species that usually have a feeding groove excavated from one side of the cell and possess one or more flagella

• Supergroup Archaeplastida = all descendants from an ancestor than engulfed cyanobacteria; all autotrophs with chloroplasts

• SAR Supergroup = very diverse group joined because of strong DNA similarity of entire genome

Amoeba overview

two subgroups = gymnamoebas (classical amoebas) & entamoebas (some are parasitic)

• move by forming pseudopods which also engulf their food

Fungi overview

• nonmotile eukaryotes

• cell wall is made of chitin

• absorptive heterotrophs → secrete exoenzymes into the environment & absorb digested nutrients

Ciliates Overview

characterized by cilia covering their outer surface that provide motility when they sweep back and forth. cilia also lines the oral groove & sweeps food particles inward to be engulfed

• paramecium & stentor

Lab 3-3 Microscopic Examination of Eukaryotic Microbes

Purpose: to observe representative samples of eukaryotes from the four subgroups and give students practice using the microscope, measuring specimens, and making wet-mount preparations.

Steps:

• if solid media: add a loopful of water and mix a loopful of specimen on the slide

• if liquid media: add a loopful of the broth directly to the slide

• lower coverglass on slide at an angle so as not to trap bubbles

• observe under microscope

Define the term pure culture

Pure culture = when a culture contains a single species.

What are the common types of media used to culture bacteria? What are the common tools used in a microbiology lab?

Media -

• Broths = used to grow microbes when fresh cultures or large numbers of cells are required

• Agar slants = used to grow stock cultures that can be refrigerated after incubation & maintained for several weeks

• Plated media = used for obtaining isolation of species, differential testing, & quantifying bacterial densities

Tools -

• inoculating loops / inoculating needles

• pipettes

• glass spreading rod

Give three reasons why it is important to practice aseptic technique in a microbiology lab

1. to prevent aerosol formation

2. to maintain a clean culture

3. to keep yourself safe

In what orientation should agar petri plates always be incubated? Why? Where on a petri plate should identification labels be written?

Agar petri plates should always be incubated upside down (lid facedown and agar up). This is so if condensation forms, it will fall onto the lid and not place moisture on the surface of the agar or interfere with microbial growth.

Identification labels should be written on the bottom of the petri dish, not on the lid. This is so that the label remains with the specimen even if the lid is lost also because the lid can rotate and ruin the orientation of the specimen on plate.

What does the term turbidity mean? Is this term used to describe the appearance of broth media, solid agar media, or both?

Turbidity = the cloudiness or opaqueness of a liquid caused by the presence of suspended particles.

• the more turbid = the more microorganisms present

Turbidity is ONLY used in broth media

Culture

a medium that contains living microbes

Aseptic transfer

Transfer without contamination

What is the most important operational risk factor for aseptic transfers?

a procedure’s potential to release microorganisms into the air as aerosols and droplets

What practices should we adhere to during aseptic transfer for our safety?

• minimize the potential of contamination (do the labs away from your book)

• be organized

• place all media tubes in a test tube rack when not in use whether they are sterile or not

• take your time

• never hold a tube culture by its cap

• hold the inoculating loop or needle like a pencil in your dominant hand and relax

When do we use a fishtail inoculation?

We use a fishtail inoculation with an agar slant culture because we are seeding the agar in a wavy pattern resembling to movement of a fish tail.

Lab 1-4 Common Aseptic Transfers and Inoculation Methods

Purpose: to introduce and practice techniques for transferring microorganisms from one medium to another without contaminating the culture or the surrounding environment.

Steps:

broth → broth

• label tube → grab new loop → flick specimen broth to ensure mixing → open cap of specimen with little finger and hold → grab one loopful of solution → replace cap → open cap of sterile broth → place inoculum in → replace cap → place inoculum in disinfectant

agar slant → agar slant

• label tube → grab new loop → open cap of specimen with little finger and hold → grab a small amount of specimen in a fishtail method → replace cap → open cap of sterile slant → place inoculum using fishtail method → replace cap → place inoculum in disinfectant

• same idea for a plate!

What does the term ubiquity mean?

Ubiquity = refers to the state of being present everywhere or in many places simultaneously

In microbiology, ubiquity often describes organisms that are widespread & found in various environments

Explain the terms mutualism, commensalism, and opportunistic pathogens

Mutualism = when a microbe actually benefits a host

Commensalism = when a microbe is benefitting but is having no significant effect on their host

Opportunistic pathogens = they are capable of producing a disease state if introduced into a suitable part of the body

What can you conclude from studying Figures 2.28 and 2.29 in your Leboffe lab manual, which feature the bacterium serratia marcescens?

The figures showcase the effect of incubation time on pigment production and the effect of incubation temperature on pigment production.

Serratia marcescens has increased pigment with a longer incubation period (ex: red after 48 hours) and has increased pigment at lower temperatures (ex: red at 25 degrees C)

Give some examples of the following categories used to describe the morphological characteristics of bacterial colonies growing on solid media → size

Size = the measurement of a colony’s dimensions

• is the diameter if the organism is circular

• is the length & width if the organism is shaped otherwise

Give some examples of the following categories used to describe the morphological characteristics of bacterial colonies growing on solid media → shape

Round (circular); Irregular; Punctiform (tiny pinpoint)

Give some examples of the following categories used to describe the morphological characteristics of bacterial colonies growing on solid media → margin

Entire (smooth); Undulate (wavy); lobate (lobed); filamentous; rhizoid (branched-like roots)

Give some examples of the following categories used to describe the morphological characteristics of bacterial colonies growing on solid media → surface

smooth; rough; wrinkled (rugose); shiny; dull

Give some examples of the following categories used to describe the morphological characteristics of bacterial colonies growing on solid media → texture

moist; mucoid (sticky); butyrous (buttery); dry

Give some examples of the following categories used to describe the morphological characteristics of bacterial colonies growing on solid media → elevation

flat; raised; convex; pulvinate (very convex); umbonate (raised in center)

Give some examples of the following categories used to describe the morphological characteristics of bacterial colonies growing on solid media → other features

color & optical properties → opaque (cannot see through) & translucent (light passes through)

What are free-living organisms?

Free-living = they do not reside on or in a specific plant or animal host and are not known to cause disease (nonpathogenic)

Saprophytes definition

decompose organic matter

What are pathogens?

If the microorganism causes damage to their host, that is, if they cause disease.

What is a reservoir?

Any area, including sites outside of the host organism, where a microbe resides and serves as a potential source of infection.

Lab 2-1 Ubiquity of Microorganisms

Purpose: To demonstrate the ubiquitous nature of microorganisms (that they are present nearly everywhere) and the ease with which many can be cultivated.

Steps: label the plates 1-8; conduct each petri dish as it pertains to the label (air, hair, vending machine, etc); if needed, use a cotton swab and gently streak the plate in a back and forth pattern; invert the plates and incubate them for 24-48 hours

What are the color, shape, and texture of microbial growth determined by?

the genetic makeup of the organism AND by environmental factors (nutrient availability, temperature, & incubation time)

How are colony morphological characteristics viewed?

With the naked eye, a hand lens, a stereo (dissecting) microscope, or a colony counter

Lab 2-2 Colony Morphology

Purpose: to observe, describe, and document the characteristics of microbial colonies growing on an agar plate.

Steps: look at cultures and compare the colonies present to the textbook. note their size, shape, margin, elevation, and texture.

Describe the basic types of morphologies and arrangements of bacterial cells

Arrangements -

• single cells → all morphologies

• diplo’s = pairs of cells → cocci & bacilli

• strepto = chain of cells → cocci & bacilli

• tetrads = group of 4 → cocci

• sarcina = cuboidal → cocci

• staphylo = irregular cluster → cocci

• palisade & angular → bacilli

Morphologies -

• cocci = sphere

• bacilli = rod

• spirilla = spirals

• vibrios = curved rods

• coccobacilli = short rods

• spirochetes = flexible spirals

Describe two important reasons for heat fixation when preparing a smear

1. adherence to the slide → heat fixation ensures that the specimen is not wasted away during the staining process

2. preservation of morphology → heat fixation kills microbes and helps preserve their size and shape, which is vital for identification

List the three basic steps in the preparation of a smear

1. cells from a culture are spread in a thin film over a small area of a microscope slide

2. the slide with specimen is air dried

3. the specimen is fixed to the slide through heat fixation (5-7 min at 60 degrees C??) or other chemical fixations

Describe the differences in the preparation technique between smear prep slides made from a liquid broth culture versus a solid agar culture

Liquid broth culture = does not need water added since it is in liquid form and can adhere as is. 2 loopfuls of broth directly onto the slide.

Solid agar culture = requires a loopful of water to be added to the slide which the microorganism from the solid media is then mixed with

Describe the potential mistakes that a novice microbiology student might make with a bacterial smear preparation?

• applying too much bacteria to the slide

• forgetting to heat fix the bacteria before staining

• not allowing the slide to dry completely before heat fixing (can burst cells or distort shape)

• not using a sterile loop

What is the key difference between a simple stain and a differential stain?

Simple stain = the use of a single dye to provide a uniform color to all cells in order to observe the size, shape, and arrangement of cells.

Differential stain = the use of 2+ dyes to distinguish between different types of cells or structures

What is the purpose of a mordant? What mordant is used for the Gram stain technique?

Mordant = a substance used in staining procedures to bind a dye to a material, making the dye less soluble. In differential staining, a mordant ensures that the primary stain will adhere effectively to the cells.

Iodine is the mordant used for a gram stain technique

What makes mycobacterium smegmatis particularly resistant to staining? Differentiate between the two methods available for acid-fast staining.

Mycobacterium smegmatis is particularly difficult to stain because it contains 60% mycolic acid in their walls (waxy layer), making them resistant to water-soluble dyes.

Ziehl-Neeson method = uses heat as part of the staining process (carbolfuchsin stain enters wall through wax by steam heating); more sensitive

Kinyoun (K) method = “cold” stain; uses a more concentrated & lipid-soluble carbolfuchsin; less sensitive

Why is heat required in the endospore stain?

Heat is required to penetrate the tough outer covering made of keratin.

Why does the endospore stain require an older culture of bacillus? What would happen if a fresh 24-hour culture were used instead?

The endospore stain requires an older culture of bacillus because the older the culture, the more endospores present. Cultures form the endospores when exposed to stress and adverse conditions.

A fresh 24-hour culture would have very few, if any, endospores present. There would be primarily vegetative cells present and the sample would be all red/pink.

Summarize the steps in the Gram staining procedure, including the reagents used and the length of time each is used. Indicate which step is the most critical in Gram staining and explain why.

Steps -

1. prepare smear prep (including the air dry & heat fixation)

2. begin with up to 3 heat-fixed emulsions on one slide and place on the staining rack

3. cover the smear with crystal violet for 1 minute

4. grasp slide with slide holder on angle and rinse with distilled water over the staining rack

5. cover the smear with gram iodine for 1 minute

6. grasp slide with slide holder on angle and rinse with distilled water over the staining rack

7. hold slide on angle and decolorize with 95% ethanol until runoff is clear, about 5-10 seconds

8. once runoff is clear, immediately rinse with distilled water

9. counterstain the smear with gram safranin for 1 minute

10. grasp slide with slide holder on an angle and rinse with distilled water over the staining rack

11. gently blot dry in a tablet of bibulous paper

12. when dry, observe under oil immersion

The most critical step in gram staining is decolorizing because prolonged exposure can remove the stain from both types of bacteria (continues to decolorize as you reach for distilled water so account for this in application time)

What would happen if a student accidentally skipped iodine during a gram stain? Effect on gram-positive bacteria? Effect on gram-negative bacteria?

Effect on gram-positive = iodine is the mordant for crystal violet so without it, the cells would not be able to hold their purple color. WOULD APPEAR PINK.

Effect on gram-negative = would still appear pink.

What would happen if a student accidentally skipped gram’s decolorizer during a gram stain? Effect on gram-positive bacteria? Effect on gram-negative bacteria?

Effect on gram-positive = they would appear purple as they are retaining crystal violet

Effect on gram-negative = they would remain purple without the decolorizing step, instead of picking up the pink tones from safranin

What would happen if a student accidentally skipped safranin during a gram stain? Effect on gram-positive bacteria? Effect on gram-negative bacteria?

Effect on gram-positive = they would still appear purple

Effect on gram-negative = they would be colorless as the decolorizer would wash away the purple from the crystal violet and no pink would be added without safranin

What would happen if a student accidentally skipped heat-fixing during a gram stain? Effect on gram-positive bacteria? Effect on gram-negative bacteria?

Effect on gram-positive = loss of heat-fixing would lead all or most cells to be washed away so the stain would be lost or barely present

Effect on gram-negative = loss of heat-fixing would lead all or most cells to be washed away so the stain would be lost or barely present

Simple Stain → what reagents are used, what is the purpose of the stain technique, and what is an example of an organism used in lab that yields POSITIVE results for this stain

Reagents -

• crystal violet = purple

• methylene blue = blue

Purpose -

• to enhance visibility of microorganisms & highlight their basic morphological features (size, shape, arrangement)

Example Organism -

• Positive result for crystal violet = bacillus cereus, E. Coli, vibrio fischeri

• Positive result for methylene blue = micrococcus luteus

Gram stain → what reagents are used, what is the purpose of the stain technique, and what is an example of an organism used in lab that yields POSITIVE results for this stain

Reagents -

• Crystal violet = purple (gram-positive)

• Iodine = mordant

• 95% ethanol decolorizer

• Safranin = pink/red (gram-negative)

Purpose -

• to classify bacteria into two main groups: gram-positive and gram-negative bacteria

Example Organism -

• Gram-positive (purple) = micrococcus luteus, bacillus cereus, corynebacterium xerosis

• Gram-negative (pink) = E. coli

Acid-Fast stain (Ziehl-Neeson) → what reagents are used, what is the purpose of the stain technique, and what is an example of an organism used in lab that yields POSITIVE results for this stain

Reagents -

• carbolfuchsin = hot pink & positive

• heat = mordant

• acid alcohol = 95% ethanol & 3% HCL

• methylene blue = blue & negative

Purpose -

• to identify acid-fast bacteria such as mycobacterium or nocardia or to determine if the bacteria present contain the waxy, mycolic acid layer within their cell wall.

Example Organisms -

• Pink (carbolfuchsin) = mycobacterium smegmatis

• Blue (methylene blue) = micrococcus luteus

Schaeffer-Fulton spore stain → what reagents are used, what is the purpose of the stain technique, and what is an example of an organism used in lab that yields POSITIVE results for this stain

Reagents -

• Malachite green = green & positive

• Heat = mordant

• Distilled water

• Safranin = pink/red & negative

Purpose -

• to differentiate between bacterial endospores and vegetative cells.

Example Organisms -

• All of the following had endospores (green) present = bacillus megaterium, bacillus subtilis, bacillus cereus, & bacillus coagulans

Capsule Stain → what reagents are used, what is the purpose of the stain technique, and what is an example of an organism used in lab that yields POSITIVE results for this stain

Reagents -

• 1% crystal violet = purple & negative (acts as the background stain)

• 20% copper sulfate = decolorizer and leaves the capsules clear or light blue

Purpose -

• to differentiate between capsule-producing cells and un-encapsulated cells

Example Organism -

• klebsiella pneumoniae

What would happen if a student performed the Schaeffer-Fulton endospore stain technique, but accidentally forgot to add safranin?

A safranin counterstain is used to stain the gram-negative vegetative cells pink. If forgotten, the vegetative cells would remain uncolored as the decolorizer has been applied, the endospore would still be green.

A student performed the endospore stain technique properly but was disappointed to discover no endospores on the smear prep slide. List two different possible explanations for this observation.

1. The culture was too young

2. The student did not add enough malachite green

3. (bonus) the steam was not hot enough / applied long enough

If a microbiology student inadvertently forgets to add the acid alcohol decolorizer to the slide during the acid-fast-staining procedure, what color would acid-fast positive and acid-fast negative cells be? Explain your answer.

The acid-fast positive and negative cells would both be hot pink because they would both retain the primary stain (carbolfuchsin) without the decolorizer.

If a microbiology student wants to determine the specific identity of an unknown bacterial culture, which staining technique would be more helpful to start with: gram stain or acid-fast stain? explain your answer.

It would be more helpful to start with a gram stain because it helps differentiate between two large groups of bacteria based off their different cell wall constituents.

What is the mordant used in the endospore stain and the acid-fast stain techniques?

Heat is the mordant used in both as it allows the dye to bypass the waxy, mycolic acid cell wall in acid-fast and bypass the keratin-covering of endospore.

Contrast (microscopy)

To be visible, the specimen must contrast with the background of the microscope field

• aided by staining

If a cell achieves a size in which the surface area is not adequate to supply the needs of its cytoplasm, what do we call this and how does the cell correct?

The surface-to-volume ratio is too small

• the cell divides its volume in half to increase surface area

What are stains?

Stains = solutions consisting of a solvent and a colored molecule

What are basic stains attracted to?

basic stains are attracted to the negative charges on the surface of most bacterial cells