Lab practical 1

describe and how to calculate resolution/resolving power

the ability to distinguish between two adjacent points, closer together the points are and still be defined as two points, the better the resolution
equation= wavelength/ 2xNA

what is numerical aperature

the number the chracterizes the range of angles over which the system can accept or emit light

what type of cleaning tissue should be used to clean the objective

lens paper

what solvent should be used hwen cleaning the objectives

ethanol/ grams decolorizer

how can you extend lamp life

dont repeatedly turn on and off, operate on low rheostat

how to calculate magnification of a microscope

magnification of ocular power x magnification of objective lens

magnification of our microscopes

4x, 10x, 40x, 100x (oil)
so magnification powers are 40, 100, 4000, 10000

function of the condenser in a microscope

concentrates light from source into a cone of light that illuminates specimen with uniform intensity

function of diaphragm

controls how much light enters the condenser below the stage

when to use the types of microscopes

bright field: morphology and external structures
darkfield: live specimen like spirochetes
phase contrast: wavelength for contrast and allows internal structures
electron: viruses and cells

what is simple staining

use of a single stain
types are positive and negative

how to smear prep

-put bacteria on plate in the middle of grease mark circle, liquid just gets put on, solid gets rubeed into water drop on plate
-use clothes pin to heat fix the bacteria to the plate by running the plate thorugh flame, allowing water to evaporate

-kills the bacteria so it isn't alive and moving
-makes the bacteria stick to slide

positve/negative staining

POSITIVE
-negative cells getting stained with positive dyes containing chromophores (color-bearing ions)
-used methylene chloride
NEGATIVE
-negatively charged chromophores that get repelled by bacterial cells, example is india ink
-looks like the negative of a picture

gram staining procedure

-crystal violet for 30 seconds
-wash with water
-mordant (grams iodine) for one minute
-wash with ethanol (decolorizer)
-wash with water
-safrinin for 1-2 minutes
-wash with water

gram staining outcomes

thick cell wall stains purple-gram positive
thin cell wall stains pink-gram negative

streak plate

4 quadrants
1-2, heat the loop, 3-4

spore staining process

- heat fix bacteria to slide
- boil the malachite green dye into the spores for 5 min
-dont let dry out, keep adding malachite green above heat
-wash off with water, counterstain with safranin to stain healthy cells.
-look under microscopes and spores will be green but healthy cells will be pink

escherichia coli

shape- bacillus
arrangement-single bacillus
gram stain- negative

staphylococcus epidermisdis

shape- coccus
arrangement- grape clusters
gram stain- positive

ALSO KNOW
-parts of microscope
-all bacterial shapes and arrangments
-cell enumeration

What type of shoes should be worn in lab?

Closed toe

When MUST goggles be worn?

When aerosolized infectious particles are potential and Bunsen burners are on.

Wgen should table tops be cleaned with disinfectant?

Before and after lab

If I wear short pants or dressed to lab, which of the following will happen?

Put on apron

When am I required to wash my hands in lab?

Before and after lab

If I remove cultures or equipment from the lab, what will be the result?

Flunk

Which microscope would you want to use in a clinical microbiology laboratory, one with a resolution of 1 nm or 100 nm.

1nm

Which light gives better resolution?

Blue

Calculate the resolution of a 100X microscope using a light source of 600 nm and a numerical aperture of 3.

600nm/(3*2)=100

Calculate the total magnification of a specimen on our microscope using a high power (40X) objective and a 10 X ocular.

400x

Which objective provides the best resolution?

64X oil, NA 1.4 better resolution not magnification

Identify one reason slides need to be heat fixed?

Kill bacteria

Identify the color-bearing ion in Methylene+ chloride-?

Methylene

Would methylene blue be classified as a positive or negative stain?

Positive

When performing a streak plate from a broth tube, which of the following instruments would you use?

Innocuolating loop

Identify the primary stain used for the Gram stain?

Crystal violet

In the Gram stain procedure, what color does a Gram negative cell stain?

Pink

In the Gram stain procedure, following the mordant step, what color does a Gram positive cell stain?

Purple

In the Gram stain procedure, following the alcohol decolorization step, what color does a Gram positive stain?

Purple

Which of the following is a characteristic of a Gram positive cell?

Thick peptidoglycan

You are Gram staining a Gram positive cell, if you leave out the mordant step what color would it stain?

Pink

Functions of glycolax

greater pathogenicity, attachment to surfaces, biofilms, source of nutrition, create a viscosity for retention of water and nutrients

precautions of capsule stain

heat fixation causes coagulation of capsule and provide false impression. capsule is water soluble. crystal violet will from crystals when air dried after washing with copper sulfate.

what media did novosphingobium capsulata grow in

litmus milk, Alkaline pH: purplish- blue color

capsule:
primary
mordant
decolorizer
results

crystal violet interacts with protein in litmus milk
copper sulfate
cells and backgrounds will be stained purple and capsules unstained

What do spores have that is unique

dipicolinic acid resistant to heat, chemical disinfectants, uv rays
must be destroyed through pressure 15 lb

SPORE STAIN:
primary
mordant
decolorizer
results

malachite
boiling
safranin
spores=green, vegetative cell=pink

what is the spore method used

Schaffer Fulton method- inoculating needle
disadvantages: messy boil, takes time
troubleshoot: must rinse with water to stain with safranin
blotting too much may rub stained smear

function of endospores

preserve dna under extreme stress
ex. mycobacterium tuberculosis

function of heat in spore

allow dye penetration

what is the gram stain method used

Hans Christian Gram

Why is a gram stain considered differential

stains 2 different colors

where can you find lipopolysaccharide

gram -

What technique is used for the acid fast

Neelson acid fast.

Acid:
primary
destain
counterstain
results

carbol fuchsin- penetrates waxy walls
acid alcohol
methylene blue
non-blue acid=pink

carbolfuchsin- 5 mins over steam bath.
water rinse- 30 secs.
acid-alcohol- 10-30 secs.
water rinse- 5 secs.
methylene blue- 2 mins.
water rinse- 30 secs.
blot dry with bibulous paper.

types of genera bacteria for acid fast

Nocardia, Mycobacterium due to mycolic acid

what species are used most for simple stain

Corynebacterium-pleomorphic and palisade arrangement
may contain metachromatic granules which function as storage sites for chemical

what type of chromophore is associated with a negative stain

negative charge

what step is omitted from staining cells

heat as it distorts size

What external bacterial cell structures can be demonstrated by a negative stain?

External bacterial cell structures such as capsules can be demonstrated by a negative stain.

Types of media solid

used for isolating colonies

Types of media semisolid

clotlike, motility and localize reaction at a specific rate

Types of media liquid

water or broth

Types of media non liquid

rice-fungi, meat-anaerobes

Types of media
gen. purpose

broad on nutrient agar or broth

Types of media
enriched

complex substances like blood, serum hemoglobin

Types of media
selective

contains 1+ agents that inhibit growth of microbes and can grow itself, feces, skin, water

Types of media
differential

variations of colonies, size, gas bubbles, preciptates Mannitol

Mannitol salt agar

considered selective and differential
selective-select for group of microbes
aureus orange
epidermis red

In the following dilution series, what is the dilution for Tube C?

1:1000

Using the same dilution series, what is the dilution factor for tube C?

1000

Using the same dilution series, if there are 56 colonies growing on Plate 3, how many colonies would you expect on plate 4?

5-6

Using the same dilution series, If there are 56 colonies growing in plate C, how many colonies would you expect on plate B?

560

Using the same dilution series and given there are 10 mls in the original inoculum, how many cells / ml would you expect in the original tube?

56000

Given the same scenario, if there are 10 mls in the original inoculum, how many total cells would there be in the original tube?

560,000

In the catalase test, what product are we looking for?

oxygen

In the urea hydrolysis test, what enzyme are we testing for?

urease

In the urea hydrolysis test, what color would a positive test turn ?

hot pink

What color would a positive citrate utilization test turn?

Prussian blue

In the mannitol fermentation test, what is indicated if an acid is produced?

yellow

In the mixed acid fermentation test, what reagent did we add?

methyl red

In the Voges Proskauer test, what color would a positive test turn?

red

What are we testing for in the oxidase test?

cytochrome oxidase

What color would a positive oxidase test turn?

purple

How quickly should an oxidase test turn to be considered positive?

20 sec

In the catalase test, what is the enzyme we are testing for?

catalase

What was the purpose of the Agarose Gel Electrophoresis?

verify the presence of PCR product

When we ran our PCR sample, did we run it to the positive (red) or negative (black) terminal?

positive

Which of the following is a true statement of electrophoresis?

smaller pieces of DNA run farther in the gel than larger pieces

Before we sent our PCR products out for sequencing, we used ExoSap-IT. What does ExoSap-It do?

hydrolyze and degrade any of the dNTPS or DNA primers remaining in sample.

Where is our DNA sequencing being done?

University of Missouri

What does PCR stand for?

Polymerase Chain Reaction

What enzyme is used in our PCR reaction?

Taq polymerase

What gene are we trying to amplify in our PCR reaction?

16s rRNA

What does the denaturation step do in the PCR reaction?

denatures DNA which will cause dna double strands to become single

What does the annealing step do in the PCR reaction?

primers bind to sequence of marker gene

What does the extension do in the PCR reaction?

new dna is added through the primers creating the complementary strands