micro midterm

**Autoclave**

uses high temperature and pressure to sterilize

List 5 safety rules

1. Tie long hair back and up
2. Never eat or drink in the laboratory
3. Disinfect lab bench before and after each lab
4. Wear safety goggles when dealing with liquids
5. Always wear a lab coat

What do you do in the event of an accident or spill?

Report it to the lab instructor or TA, notify students, cover with paper towel and saturate with bleach for 20 minutes. Clean area with disinfectant and place paper towels in reservoir. 

**Transient bacteria**

colonize superficial layers of skin, easier to remove with handwashing, most frequently associated with health-care-related infections

**resident bacteria**

part of normal flora, attached to deeper layers of skin, more resistant to removal

**Opportunistic infections**:

contributed to by transient and resident flora, occur in people with compromised immune systems

**Nosocomial infections**:

acquired in a healthcare setting, associated with transient flora

What are universal precautions? Why are they important? On what body fluids do these precautions apply?

A set of guidelines to reduce the risk of disease transmission. They help keep people safe in healthcare settings. They apply for blood, semen, other fluids

**TSA**:

tryptic soy agar, all-purpose medium that does not support the growth of fastidious organisms

**Fastidious organisms**:

organisms with complex nutritional needs

Compare and contrast Semmelweis and Lister

* Semmelweis: Implemented policy of physicians washing hands with chlorine of lime, ridiculed and was killed by streptococcus
* Lister: implemented aseptic procedures initiated by Semmelweis, proved effectiveness of handwashing in decreasing surgical wound infection and won award

List 3 commonly used agents for handwashing. Which is most effective?

* Washing 
* Washing and scrubbing
* Alcohol gel (most effective)

Describe proper handwashing

* Wash hands with warm water and soap for 30 seconds
* Turn off faucet with paper towel

List universal precautions

* Wash hands before and after patient contact and change gloves between each patient 
* Wear gloves and gowns if soiling of hands, exposed skin, or clothing with blood or body fluids is likely
* Wear masks and protective eyewear or chin-length plastic face shields whenever splashing or splattering of blood or body fluids was likely
* Use disposable mouthpiece/airway for cardio-pulmonary resuscitation
* Discard contaminated needles and other sharp objects in a “sharps” container. Needles must not be bent, clipped, or recapped
* Clean up spills of blood or contaminated fluids by putting on gloves, wiping up with towels, washing with soap and disinfecting with 1:10 solution of bleach and water

Give 3 methods for reducing the dissemination of Staph aureus from patient to staff, staff to patient, and patient to patient

1. Wear gloves when touching patients
2.  Wash hands and use alcohol gels, wear gloves and change often
3. Change gloves between patients, change materials between patients

Distinguish between sterilization, disinfection, and antisepsis

* Sterilization: The complete destruction of all living material including endospores
* Disinfection: destruction of vegetative organisms, but not endospores, on inanimate surfaces or object
* Antisepsis: the process of making something contamination-free to where it would not reproduce or create harmful microorganisms

List two advantages of handwashing over gloves. List two advantages of gloves over handwashing

1. Handwashing over gloves: gloves may not be changed as often as hands would be washed, disinfecting with alcohol can be very effective for reducing bacteria
2. Gloves over handwashing: gloves keep the person from touching the patient skin to skin, gloves can be taken off and sterile ones put on instead of washing

**Resolving power**

the ability to distinguish two very small or closely spaced objects

**Parfocal**

lens are adjusted so that specimen remains almost in focus after objective rotates

**numerical aperture**

a mathematical constant that describes the relative efficiency of a lens in bending light rays

**Wet mount**

the observation of particulate objects or living organisms in an aqueous solution

**Buds**

new yeast cells that form from parent cells

**Dimorphic**

fungi that are yeasts at 37 C and moldlike at 25 C

**Pseudohyphae**

buds that fail to detach and form short chains

**Deuteromycetes**

reproduce solely asexually

**Superficial mycoses**

cause infections of skin and hair

**Cutaneous mycoses**

involve nails 

**Subcutaneous mycoses**

involve deeper layers of skin

**Systemic mycoses**

cause infections of subcutaneous and deeper tissues such as nervous systems, lungs and genitals

**Molds**

multicellular filamentous fungi

**Thallus**:

a macroscopic mold colony

**Mycelia**:

mat of strands that compose a thallus

**Hyphae**:

individual filaments within mycelia

**Vegetative hyphae**

grow on surface of growth medium

**Aerial hyphae:**

rise from the surface of growth medium

**Reproductive hyphae:**

aerial hyphae that give rise to spores

**Septum**

individual cells separated by cross walls that make up hyphal strands

**Septate**

hyphae made up of septums

**Coenocytic hyphae**

fungi that lack cross walls and are continuous mass of cytoplasm containing multiple nuclei

**Potato dextrose sugar/sab-dex**

simple in composition, low pH, inhibit bacterial growth

**Lacto-phenol cotton blue**

helps visualize hyphal and reproductive structures

**Trophozoites**

active stage of some parasites, growing and getting nutrients

**Cysts**

dormant stage of some parasites

How does the inclusion of a blue light filter change resolution?

It cuts out the longer wavelengths of light so that the shorter ones are left, increasing resolution

What is Brownian movement?

The random motion of particles suspended in a medium

Why is immersion oil important on the oil immersion lens?

The oil keeps the light from scattering and instead sends it right into the lens

Equations for total magnification and limit of resolution

* Total magnification: objective x ocular
* Limit of resolution: wavelength/2x numerical aperture

List guidelines for how to use and put away the microscope

* Always start as close as possible and move away
* Remove oil 
* Place low objective in light path
* Always carry with both hands

List 3 categories of eukaryotic microbes

* Fungi
* Parasitic protozoa
* Parasitic helminths

List 3 characteristics for identifying fungi

* Color of colony
* Size of colony
* Fluorescence 

List 4 major classes of medically important protozoa and give examples

* Amoebas: *Entamoeba histolytica*
* Flagellates: *Giardia lamblia*
* Ciliates: *Balantidium coli*
* Sporozoa: *Plasmodium falciparum*

Compare and contrast parasitic worm eggs

* *A. lumbricoides* eggs: larger, embryonated, surface covered by small bumps
* *E. vermicularis*: smaller, flattened on one side

**Bergey’s Manual of Determinative Bacteriology**

**taxonomically classifies and documents most known bacteria** 

**Gracilicutes**

one of major groups of bacteria; “thin skinned”, Gram negative

**Firmicutes:**

one of major groups of bacteria; “thick skinned”, Gram positive

**Tenericutes**

one of major groups of bacteria; “soft skin”, no cell wall

**Mendosicutes**

one of major groups of bacteria; archaea

**Genus**:

first part of scientific name, can be shortened to only the first letter

**Specific epithet:**

species, written out

**Strains/types/vars**

differences within a bacterial species

**Biotypes**

differ biochemically

morphotypes

differ morphologically

serotypes

differ serologically

List and describe biosafety levels

* Class 1: unlikely to cause human disease 
* Class 2: can cause human disease and hazard to workers, unlikely to spread to community, effective treatment available
* Class 3: causes severe human disease, presents a serious hazard to workers, presents risk to community but usually effective treatment
* Class 4: causes severe human disease, severe threat to workers, high risk to community, no effective treatment

List 5 techniques used in identification of bacterial species

* Morphology
* Serological profiles
* Biochemical tests
* Staining characteristics
* Fatty acid profiles

List 5 basic rules to follow when performing aseptic technique

* Always sit down with both feet in footwell
* Always flame lip of tube before and after loop
* Never work over your lap
* Allow loop to cool before entering medium
* Never place tube cap on work surface or touch it to anything

List 3 rules to follow when working with pipettes

* Do not flame pipette tip
* Always wear goggles when working with broth
* Do not invert pipette after liquid has been drawn up

Define pure culture, contamination, and aerosol

* **pure culture**: completely sterile culture
* **contamination**: causing a sterile object to no longer be sterile
* **aerosol**: bacteria in the air

Differentiate between a mixed culture and a contaminated culture

* **mixed culture**: culture growing 2 or more known microbial species
* **contaminated culture**: culture that was once pure but now contains unwanted species

Give 2 important reasons for aseptic technique

* preventing contamination of cultures you are trying to isolate
* preventing contamination of yourself or others

What do you do if you spill a culture?

* minor: cover with paper towel, soak with bleach, let sit for 20 mins, put in disinfectant beaker and wash hands
* major: tell someone, clear lab, change clothes

**simple stains**

used principally for morphological studies

**Differential stains**

used to differentiate cells, often based on differences in cell wall structure (gram and acid-fast)

**Special stains**

used to highlight structural components of the bacterial cell (flagella and capsule stains)

Distinguish between acidic and basic stains and positive and negative stains

* **Acidic**: contain chromophores that are negatively charged and are repelled by bacteria, background stained while cells are colorless (negative stain) (nigrosin)
* **Basic**: chromophores are positively charged and are attracted to cell surface; used in simple and differential stains(crystal violet)
* **Positive**: bacteria is colored, background is white
* **Negative**: background is colored, bacteria is colorless

Contrast the procedure for making a slide from a broth and making a slide from a slant. Which one is best for determining arrangement?

* Slide from broth: place water drop on slide, put bacteria on with loop, dry, fix, stain (better for arrangements)
* Slide from slant: place water drop on slide, put bacteria in with pipette, dry, fix, stain

What would a gram-positive smear look like that was halted after the decolorization step? Gram-negative smear?

* Gram-positive: purple
* Gram negative: colorless

What 2 factors may make Gram-positive bacteria appear Gram-negative?

* cells are old and do not hold stain
* prolonged exposure to alcohol

What factor could make Gram-negative bacteria appear Gram-positive?

clumping of dye or staining of something that is not bacteria

What is a mordant? What reagent acts as a mordant in the Gram reaction?

Something that forms a complex with the dye, Gram’s iodine

Why does Gram-positive smear stain purple and the Gram negative smear stain red?

The Gram-negative outer membrane and thin peptidoglycan layer can be dissolved by alcohol. This removes the purple dye from the gram-negative bacteria and then saffranin stains it red. The Gram-positive cell wall cannot be broken down by alcohol and therefore doesn’t lose the purple stain

List the process of doing a Gram Stain

* Prepare smear
* Crystal violet(30 sec) and rinse
* Gram’s iodine(1 min) and rinse
* Apply acetate alcohol until no more crystal violet flows off
* Immediately rinse with water
* Apply safranin(1 min) and rinse

List some common mistakes in preparing a Gram stain

* Too much bacteria
* Overheating smear
* Rinsing too long
* Not timing decolorizing agent right
* Using old cells
* Confusing crystals for cells

**Zeihl-Neelsen stain**

acid-fast stain that distinguishes between Mycobacterium and Nocardia and other species

**Acid-fast**

bacteria that take up dye but don’t lose it

List organisms considered acid-fast

* *Mycobacterium*
* *Nocardia*
* *Cryptosporidium*

What color is AFP? AFN?

* AFP: fuschia
* AFN: colorless, stains blue

What method of testing has replaced acid-fast stain in developed countries?

Fluorescence staining

What are the distinguishing features of Mycobacterium cell wall?

Thick mycolic acid lipid layer makes it acid-fast

**Endospore stain**

structural stain that stains endospores green and vegetative cells red

**Heat shock method**

method for the detection of small quantities of spores, kills vegetative cells but not endospores

List genera that are endospore formers

* Bacillus
* Clostridium

What chemical components of endospores render them resistant to heat?

Dipicolonic acid and calcium in the surrounding cap

List 3 methods for determining motility. Which is the hardest? Which is most misinterpreted?

* Flagella stain(hardest)
* Hanging drop
* Semisolid media(most misinterpreted)

Define positive and negative chemotaxis

* Positive: movement towards substance
* Negative: movement away from substance

What does “swarmers” mean?

The cells migrate rapidly over surfaces, forming whirls

**Obligate aerobes:**

absolutely dependent on oxygen

**Facultative anaerobes**:

grow in presence or absence of oxygen

**Microaerophiles**:

grow in small oxygen levels

**Candle jar:**

obtain gas levels