Microbio Lab Midterm

Lab Attire?

Lab coats, gloves, goggles, long pants, closed toed shoes, tie back hair

Directions after a spill

Let a TA and ppl around you know, dont clean yourself

Sharps location

Provided, located at TA desk in front of lab
Dont clean yourself

First aid location

located in hallway

Lab safety

Pull handle stand under shower till flow stops

Eye wash station

push handle, lower face until eyes are in flow. Keep lids open and all water to rinse

Food?

No food cough drops drinks phones

Cleaning lab space

Wipe down with 70% EtOH

Wipe down with Bleach after

Disposal of Test tubes

Round baskets provided

Disposal of Petri dishes

Provided disposal trays

Disposal of Flasks

On table where petridishes and testtubes will be placed

Dispsal of pipettes

Round plastic containers at lab bench end, tips down

Glove disposal

Large red glove bins labeled gloves

Labeling petri dishes

Bottom of plate

Write seat#, name, date, and organism innoculated

Storage of petri dishes and why, temp?

Bottom on top, agar side up

PRevent drippy condensation

37 degrees celsius incubation

Common test tube preperations

Broth

Agar slant

Agar deep tube

Handling test tubes

Do not carry by the cap

Carry in a rack

Hold in non dominant hand

remove caps with dominant hand

Labelling

Label on clear tub portion

Dont label caps

China markers

Inoculating loop handling

USed to transfer culture

Sterilized before and after use

Microincinerator use

Sterilization of loops and test tubes

Plug in at beginning of each lab

Only touch base of each barrel

Sterilizing the loop steps

Insert loop up to handle

Do not rest against inside of incinerator

¾ of loop should glow hot

Remove and cool

Asepsis definition

Without infection

Steps of two tube transfer

Resuspend broth

1. Sterilize loop

2. Uncap tubes

3. Sterilize tubes

4. Transfer from culture to fresh medium

5. sterilize tubes

6. recap tubes

7. sterilize loops

Reasons for staining

Provide contrast

IDentify bacterial morphology size and arrangement

Simple stain

Also known as cationic stain

Useful for distinguishing different morphologies, arrangement, and relative sizes

Colors the cells and provides contrast under bright field

Positive charge of the dye interacts with negative charge of bacterial cell wall (colors cell surface)

Cationic stain example of how it works

Crystal violet (+) also methylene blue ion sticks to negative charge of bacterial cell wall

Negative Stain

Stain doesn’t penetrate cell

• Negatively charged stain repelled by negative charge at cell surface

• Cell appears transparent

Examine morphology and size

• Cells are not heat fixed

USeful for organisms that do not stain easily

• Nigrosin- black anionic dye (what we will be using)

  • REpelled by negatively charged bacteria cell surface

Smear preperation from broth

Aseptically transfer 1 loopful of culture to a clean slide and sread to maximum thinness

Allow to air dry

Heat fix by holding over microincinerator for 10 seconds (clothespin)

Smear prep from slant/plat

Add 1 loopful of water to slide first

Aseptically collect bacterial sample from plate or slant

• Just touch the slant/plate, then mix with water on slide

Allow to air dry

Heat fix by holding over microincinerator for 10 seconds with clothespin

Microscope storage

Clean oil off 100x objective using lens cleaner and kimtech wipe

Lower stage as low as possible

Move the 4x objective into position

Make sure cord is properly wrapped up

Bright field microscopy principles

1. Illumination

2. Magnification

3. Resolution

Illumination definition

Light source

Condensor: directs light towards the objective lens in bright field microscopy

Iris diaphragm: adjusts the diameter of the cone of light so that it just fills the objective lens

Magnification

Ocular: 10X

Objectives:4X, 10X, 40X, 100X

• Ocular x objective=total magnification

Resolution

Resolution is the smallest distance between two objects which can be seen as seperate

Our scopes resolving power ~0.2um while the average microbe is 1.0um

Oil immersion

100X requires immersion

refractive index of oil is that of glass

Resolving power calculation

Resolution=wavelength/(2xNumericalAperture)

d=lambda/(2NA)

Numerical aprture

Light gethering capacity

Wavelength

illumination (nm)

Cocci

Spheres

Diplococci

di(two) spheres

Streptococci

Chaine spheres

Sarcinae

Or tetrads, 4 spheres

Staphylococci

Clumps of spheres

BAcilli

Rod shaped

Streptobacilli

Chain rods

Spirochetes

Coil

Vibrios

Curved bacilli with flagella

Coccobacilli

Cocci and bacillus

Differential stain

Detects differences between organisms through the use of many reagents

Simple stain

One dye used stains all cells present

Structural stain

Confirm structural characteristics of cell

Gram stain

Developed by a Hans Christian Gram in mid 1880s (dane)

Looking to find the bacterial cause for pneumonia

USed crystal violet and alcohol wash, found some would retain purple color

He considered it a failure

Gram-positive cells

Thick layer of petptidoglycan, which retains crystal violet even after ethanolw ash

Gram-negative

Thin layer of peptidoglycan, does not retain crystal violet after ethanol wash

Reagents of gram stain

Crystal violet- primary dye

Gram’s iodine- mordant

95% EtOH- decolorizer

Safranin- secondary dye, counter stain

Gram stain procedure

Smear prep

1) apply crystal violet (1 minute)

2) Wash with DI Water

3) Apply grams iodine sit for 1 minute

4)rinse with EtOH for 10 seconds

5) apply safranin and allow to sit for 1 minute

6) Rinse with DI water

Color of gram positive?

Purple (crystal violet)

Gram negative

Pink or red

Function of EtOH in gram stain?

Differentially removes Crystal violet from gram negative

Acid fast Microorganism

Organisms with a high mycolic acid content in cell walls resist staining

Steam used to penetrate cell wall

FAstness is ability to retain dye when treated with a decolorizer

Reagents of an acid fast stain

Carbol fuchsin- primary dye

• steam is used to insert carbol fuchsin

Acid alcohol- is a decolorizer

Methylene blue- is the secondary dye or counterstain

Procedure for acid fast

1. Steam w carbol fuchsin for 3 minutes

2. decolorize with acid alcohol for 10 seconds

3. counterstain with methylene blue

Acid fast stained cells color

Cerise or pink/violet stained with carbol fuchsin

Non acid fast stained cells color

Blue, stained with methylene blue

Acid fast genera

Mycobacterium tuberculosis causes tuberculosis

Mycobacteria leprae- hansens disease (leprosy)

Spore staining

Schaeffer-Fulton method, structural stain that can be used to detect dormant forms of bacteria- endospores

Can be released, free spores

Force primary dye into resistant endospore via steaming

Stained spores are then resistant to decolorizer

Reagents of spore staining

Malachite green- primary dye

• Steamed

Water- decolorizer

Safranin- secondary dye or counter stain

Procedure

1. Steam w malachite green for 3 minutes

2. Decolorize with water

3. Counter stain with safranin

Endospore identification

Spores are green inside pink bacterial cell

Free spore identification

Small green oval body

Just a cell

Will be pink

Genera of spore formers

Bacillus: gram-positive rod (bacillus anthracis)

Clostridium: gram positive rods (C. botulinium, tetani, difficile)

Sporosarcina

Pure culture

Contains a single microbial species

Environmental and patient samples always contain a mix of microbes

Pure culture are important in both a clinical and research setting

Pure culture techniques aim to dilute bacterial samples in order to seperate individual cells that can grow into isolated colonies when plated

Types of growth medium

General purpose

Selective

Differential

General purpose

Support growth of organism

Often used to try to isolate microbes from a mixed culture or grow microbes

Solidifying agent use for pure culture

Agar

Solid media is preferred and microbes cannot metabolize agar

Pour plate method

May be quantitative or non-quantitative depending on dilution method

Culture serial diluted in molten agar >40 degrees celsius and then plated

Colonies grow through out the plate

Spread plate

Seriel dilution with broth culture

can be quantitative or non quantitative

Small sample is diluted and spread across surface of plate

Colonies on surface

Streak Plate technique

3 Zone
Isolation zone streak method

3 tiered zones (I, II, III) final zone IIIB

Method used to seperate a single species from a mixed population

Sample plated across 3 zones

Primary streak

A three zone streak plated with a mixed culture, used two seperate microorganism into colonies

Secondary streak plate

Subculture from one of the colonies found from primary streak

A pure culture

General

Includes: Nutrient Agar (NA), Tryptic Soy Agar (TSA), Brain HEart Infusion (BHI)

Selective media

Use inhibitors to prevent growth of certain organisms

Selective media examples

Phenyl Ethyl Agar (PEA), Eosin Methylene Blue (EMB), MacConkey Agar (MAC)

Differential media

allow growth of many microbes but differentiation is seen by indicators that detect change that have occured

Dye, reagents, blood cells, culture conditions

Differential media examples

Eosin Methylene blue, blood agar, MacConkey Agar

Combination media

BOth selective and differential

Combination media examples

EMB and MacConkey

Complex media definitions

Undefined, exact chemical comp is not know and nutrients originate from extracts or digests from natural sources

BHI and TSA

Defined media

Known chemical composition for each component

Phenylethyl Alcohol

PEA

Only selective

Prevents gram negative growth

USed to select for gram positive,

• Staphylococci

• Gets rid of E. coli

Blood Agar

BA

Differential Only

Indicator: red blood cells

• TSA base with 5% sheep’s red blood cells added

Blood agar can be useful in differentiating hemolysis patterns of microorganisms

• Lysis of RBCs

Different types of hemolysis

• BEta

• Alpha

• Gamma

Beta Hemolysis

Best, complete

Alpha hemolysis

Alright, average

partial hemolysis

Gamma hemolysis

GArbage, no hemolysis

MacConkey Agar

Selective and differential- combination

Used for identification of gram negative enterics

• Enterobacteriaceae

Mac Indicators

Neutral red,a dye a pH indicator

Differentiate lactose fermenters from non fermenters

Lactose fermenters create a hot pink color

No fermentation results in no pink color change

Mac Inhibitors

Crystal violet and bile salts

Prevent gram positive growth

Eosin Methylene Bue

EMB

selective and differential- combination

USed to search form coliforms

• fecal coliforms

  • Also can be used to test for UTIs

EMB Inhibitors

Eosin and methylene

• Prevents growth of gram positives