CBU BIO205L Microbio Lab Practicum 1

micrococcus luteus looks

gram+

rod

staphylococcus

gram + spherical organisms about 1 micrometer in diameter; salt tolerant

staphylococcus aureus characteristics

• medium large, raised colonies on blood sugar, cream/golden yellow color on naladixic acid agar

• gram+, cocci in clusters

• beta-hemolytic on sheep blood agar

• positive for clumping

• positive for free coagulase

• growth and fermentation MSA

• positive for DNase

escherichia coli looks

gram -

rod/enteric bacteria

bacillus subtilis looks

gram +

rod

isolation of staphylococci purpose

to isolate and identify staphylococci species from the nasal cavity and other environments

klebsiella pneumoniae

gram -

rod shaped

bacillus cereus looks

gram +

rod

slime layer

thin and flowing capsules

Culture

one or more species and it involves nutrients growth and temperature

mixed culture

microbial culture consisting of two or more species

culture medium

composition of nutrients

Nutrients Broth

a liquid solution, involves bacteria and protists

sterilized

All living organisms and viruses are killed and no spores exist.

culture tubes

used to hold specimen and or other fluids to be taken for further testing.

culture plates / culture dishes

cultivate microorganism

subculturing

transferring microorganisms from one medium to another

Subculturing/Aseptic conditions

Transfer between nutrient media without contamination with other microorganisms.

inoculating loops and needles

materials used to perform a transfer

Bunsen burner

used to heat substances/incineration

pure culture

contains only one species or strain of bacterium

agar

solidifying agent, a polysaccharide extracted from seaweed (red algae)

Basic Growth Media

• to grow solution w/o agar: liquid media - broth - maintaining pure cultures

• to grow solution w/ agar: - solid media - agar slants (pure cultures/physiological studies), agar deep (physiological studies) or agar plates ( isolation of pure cultures and study of microbial diversity)

Subculture steps

1. loop sterilization process

2. transfer process

3. capping tubes abd re-sterilize the loop

pipettes

material used to transfer liquid

incubator

is a type of fridge to store specimens

water bath

used for holding test tubes or slides which must be heated

streak plate

used to separate and isolate bacterial cells in mixed population

colonies

cluster of cells derived through multiplication from single cells/spores

subculture transfer purpose

transfer aseptically a pure culture of bacteria from culture tube to another sterile tube

streak plate purpose

isolating a single bacterial species in pure culture

Quadrant Streak Method

relatively inexpensive and straight foward method for separating bacterial cells in mixed populations of high cell density

streak plate steps

1. streak loopful bacteria in one area of agar plate

2. flame loop and streak bacteria from 1st to 2nd area

3. flame loop and streak bacteria from 2nd to 3rd area

4. dont flame loop and streak bacteria from 3rd to 4th

magnification

increasing the apparent size of the specimen being observed

working distance

refers to the distance between the slide and the bottom of objective lens

resolving power

determines the size of the smallest object that can be seen clearly under specified conditions

field

single circular area (what you're looking at when using microscope)

refractive index

pertain to the light-bending ability of glass, oil, and air materials through which light must pass image formation

parts of microscope purpose

to identify parts of microscope and explain their functions by focusing on a sample specimen

parfocal

object in view under one objective lens will still be in view under another lens

microscope examination purpose

to observe specimens w/ the oil immersion objective lens

1. object is observed clearly under low power, but you lose it when moving to high power

place the object in the center

2. black half-moon or quarter-moon off to the side

clicked position

3. object at 400x but cannot be found when you move to oil immersion

rotate the nosepiece forward to the lower objective

4. cannot focus down to level where specimen is

check if slide not upside down

5. encounter a fuzzy image under high power

moisten lens tissue with lens cleaner and clean the objective

6. image is too dark to see

open diaphramph to let light in

7. cleaned ocular lens thoroughly and image not clear

may be layer of oil on lens of condenser

8. lint and debris you see are on the slide or in the lens system

slowly move the position of stage knobs

9. should wear glasses while using the microscope

adjusted for the viewers vision and glasses

10. binocular microscope and you have trouble focusing with both eyes open

check oculars to see which one has an ocular adjustment

microscope steps

• place slide on center stage

• focus at 10x

• center in field and rotate to 40x

• use fine focus to resolve and center

bacterial smear purpose

to prepare bacterial sample for staining

heat fix

kills and adheres bacteria to slide

slide label

naming the bacterial species

bacterial smear steps

• label slide

• sterilize inoculating loop

• remove loopful bacteria

• smear broth on target area

• air dry and heat fix

basic stain

positively charged stain, the use of the positively charged stain allows it to bind the negatively charge cell wall, thus staining the cell.

simple stain

one stain used

simple stain purpose

determine size shape and arrangement of bacterial cells

simple stain steps

• stain heat fixed slide w/ methylene blue

• wash and dry

• observe

negative stain

a simple stain that stains the background but leaves the bacteria unstained

negative stain purpose

accurately measure cell size and determine cell shape in an undistorted manner

negative stain steps

• loopful bacteria at east end

• drop of nigrosin on top

• smear at angle towards west side

• air dry

mixed population

bacteria usually exists in these, soil, water, and some human body parts

prepared slides

microscope slides that have dead bacteria and often stained slides ready to be viewed

selective media

used to isolate specific groups of bacteria growing on agar

differential media

facilitate a visual differentiation of different species of bacterial colonies growing on an agar plate

enteric bacteria

found in animal/human intestinal tract are gram negative non spore forming rods

- can be isolated from fecal samples

-consist of large number of genera that live within or are responsible for infections of the intestinal tract

-cause typhoid fever, shigellosis and travelers diarrhea

clostridium

consists of sporeforming species but it can be separated from bacillus by its necessity for growth under anaerobic conditions

EMB agar

- EMB (Levine eosin methylene blue)

-differential medium, doesn't support growth of gram+

-pigmented colonies formed by lactose fermenters, pigmentation is distinctive to various genera

MacConkey Agar

- differential medium containing bile salts to inhibit non enteric bacteria (has 2 dyes: neutral red/crystal violet which are taken up by lactose fermenting bacteria

- CV: inhibitory to the growth of gram+

- Enteric bacteria form pigmented colonies while the non lactose fermenters form colorless colonies

(Red colonies = positive lactose fermentation

Colorless colonies = negative lactose fermentation)

MacConkey Agar color results for lactose fermenting bacteria

Escherichia coli- brick red

Enterobacter/Klebsiella & other lactose fermenters - pink to red with a mucoid texture

what disease does staphylococcus aureus cause?

toxic shock syndrome, food poisoning, boils

gram negative stains what color

stains pink/red

mannitol salt agar

medium that is both selective and differential; salt concentration is a high 7% to inhibit other organisms and available for fermentation of acid

why can staph be found on ham?

bc s. aureus ferments in mannitol

gram positive stains what color

(keep your P's together, Positive = purple)

stains purple

what disease does staphylococcus saprophyticus cause?

urinary tract infections UTI

Endospores

gram postive bacterial genera, (bacillus and clostridium)

gram stain purpose

to differentiate between gram-positive and gram-negative bacteria to determine cell size, shape, and arrangement

gram stain steps

1 air dry & heat fix bacteria

2 flood smears with crystal violet for 1min

3 rinse with water

4 cover smear with Gram's iodine for 1min

5 rinse with water

6 decolonize with 95% ethyl alcohol for 15 sec (twice)

7 rinse with water

8 counter stain with safranin for 1min

9 rinse smear with water

10 blot dry with bibulous paper

Gram positive info

-Bacteria that KEEPS color after using alcohol are: gram-positive

-Gram-pos have large amounts of peptidoglycan so they "trap" stain well

-Gram-pos result: remains purplish-blue

spore stain steps

1 air dry heat fix smear

2 fill beaker with water on top of burner & allow to boil (ensuring there is steam)

3 place wire pad ontop of beaker

4 put blotting paper ontop of slide big enough to cover bacterial smear

5 place slide (with paper) ontop of wire pad

6 flood malachite green onto paper for 3min DO NOT LET DRY OR POUR OVER

7 remove slide & remove paper

8 allow it to cool then rinse with water

9 flood smear with safranin for 1min

10 rinse smear with water

11 blot smear

gram stain technique

the most important differential staining procedure in microbio bc the great majority of bac are either gram+ or gram-

enriched media

where addtional growth factors are added to a medium, also can act as a selective or differential medium

who discovered gram staining

Christian Gram

species of staphylococcus are tolerant to....

salt, therefore can be selected out from a mixture of bacteria in a high salt medium

spirochetes and mycobacteria

what are the two exceptions to gram stain reactions

spore stain purpose

to view and contrast vegatative cells from endospores with the microscope

capsule stain purpose

to detect the presence or absence of a bacterial capsule

capsule stain steps

1 prepare negative stain

2 allow to air dry

3 flood slide with crystal violet for 1min

4 wash excess stain with 20% copper sulfate

5 blot gently

what bacteria is used for simple stain?

staphylococcus aureus

bacillus subtilis

what bacteria is used for negative stains?

capsule

a layer of polysaccharides and proteins secreted by certain bacteria including many pathogens

capsules protect what

protects the bacterium against dehydration and traps nutrients from the surrounding environment

why capsule stain may be illegible

heat fixing and using water instead of saline to rinse, or using too much saline

difference between negative staining and CV staining

negative: stains the background to outline the capsule

CV: stains the cells

are capsules easily destroyed by water and heat

yes

what stains are heat fixed

simple stain, gram stain, spore stain

methylene blue

stain used for simple stain