CSN 251 Lab practical #1

differential media

show differences between microbial colonies based on their cultural characteristics (what they look like when growing in colonies)

ubiquitous

they can be found in every habitat on earth

microorganisms

the most numerous and most diverse life forms on earth

symbiotic relationship

close interaction between species in which one species lives in or on the other

commensals

microbes that inhabit our bodies in ways that may benefit both them and us

opportunists

Microbes that cause disease in the right circumstances. When they have the opportunity to cause a disease they will often do so.

fomite

a person or object that mechanically carries microbes from one person or place to another

culture medium

used as a nutrient source to allow microbes to grow

liquid culture medium

liquid at room temperature

solid culture medium

solid at room temperature; typically made of agar

petri plates

agar solid media in round plastic culture plates with loose fitting lids

inoculating

introducing microorganisms into a culture where they can grow and reproduce

general purpose media

allow the growth of many microorganisms that do not need special growth factors

enrichment media

contain special growth factors required by some microorganisms to grow; i.e. blood agar

growth factors

stimulate the growth and division of cells

selective media

inhibit the growth of some groups of microorganisms and select for the growth of other microorganisms

hemolysis

lysis of blood cells

tryptic soy agar

general or all purpose culture media plate (a)

sheep blood agar

enrichment and differential media plate (b)

colony

clones from one bacterium

TSA

tryptic soy agar

SDA

Sabouraud's agar

BAP

blood agar plate

colonies

individual clustered groups of visible growth

cultural characteristics

size, shape and color can help in the identification of unknown microorganisms

Environmental samples

TSA and SDA

alpha hemolysis

partial hemolysis of red blood cells

gamma hemolysis

not hemolytic

normal microbiota

microbes normally present in and on the human body

beta hemolysis

complete hemolysis

light microscopy

brightfield microscopy produces an image by transmitting light through a specimen

ocular lens

microscope part (A)

objectives

microscope part (B)

arm

microscope part (C)

condenser lens and adjustment knob

microscope part (D)

coarse adjustment knob

microscope part (E)

fine adjustment knob

microscope part (F)

lamp switch

microscope part (G)

base

microscope part (H)

rheostat

microscope part (I)

mechanical stage

microscope part (J)

4x

scanning objective

10x

low power objective

40x

high dry objective

100x

oil immersion objective

domains

tree of life classifications

Archaea, Bacteria, Eukarya

What are the three domains?

protist, fungi, plants, animals

Domain Eukarya subdivided kingdoms

prokaryotic cells

relatively small and simple in structure and have generally have no true nucleus

eukaryotic cells

typically larger and more complex than prokaryotic cells. They contain a nucleus and other membrane bound organelles

unicellular

made of a single cell

multicellular

consisting of many cells

colonial and filamentous forms

protists that are intermediate kinds of organization

yeasts

Kingdom Fungi's unicellular microscopic members

molds

Kingdom Fungi's multicellular but are microscopic and macroscopic

protozoa

protist

locomotion

movement

algal protists

unicellular, free-living eukaryotes, although some __________ are filamentous and colonial

producers

photosynthetic phytoplankton and consumers

heterotrophic decomposers

use organics for carbon and energy

mycelium

mass of hyphae

budding

Asexual reproduction in which a part of the parent organism pinches off and forms a new organism

spores

Asexual reproductive or resting cell capable of developing into a new organism without fusion with another cell, in contrast to a gamete

Aseptic technique

working with microbial cultures that insures the environment, personnel, and the microbial cultures are NOT contaminated

pathogens

microbes that cause disease

Work Area Disinfection

The work area (lab bench) is treated with disinfectant twice when working in the lab. The first disinfection is performed when first entering the lab prior to any materials being placed on the bench. The disinfection will destroy most vegetative cells and viruses but generally will not destroy endospores. Therefore it is not a method of sterilization. The physical process of scrubbing the bench removes microorganisms. The work area is also disinfected after work is complete.

Bacteriological Loops and Needles

The transfers of culture material are achieved by using inoculating loops and needles which are sterilized before and after coming in contact with the culture. The incinerator is used to sterilize the loop or needle by placing in the incinerator opening until they glow orange. The loop or needle must be cooled prior to use which is done by holding for at least 5 seconds. The loop or needle is sterilized again after transferring cultures and before returning to the bench to prevent contamination of the environment. Do not store the loop or needle in the incinerator as the loop becomes so hot the rubber holder may melt. Return the loop or needle to the bench top location.

Culture Tube Handling, Flaming, and Inoculation

Hold culture tubes in your non-dominant hand. Prior to inserting a sterile, cooled loop or needle into the culture, the cap is removed, and the mouth is held next to the incinerator opening. Pass the culture next to the hot opening several times to produce convection currents to prevent microbes from the air from entering the culture. Hold the broth tube at an angle but still upright to minimize the chance of airborne contamination. When finished inoculating, flame the culture mouth again using the same procedure, and replace the cap on the tube. The broth tube is placed in a test tube rack and not laid on the bench top.

Petri Plate Inoculations

The plate lid is used as a shield to prevent airborne contamination by raising the cover and holding it diagonally over the plate. The loop with the inoculum is streaked over the agar surface gently to as the agar can be torn or gouged. The cover is replaced and loop is flamed.

Hand Washing (degerming).

Hands are washed as the last act before students leave the lab and after the bench top disinfection. Students also wash hands if they come into contact with bacterial cultures

Minimize Potential of Contamination

Do not perform transfers of microbes over your books and papers. Place materials safely away from the culture material

Suspend Bacteria in a Broth

Mix broth cultures gently prior to obtaining inoculum by drumming your

fingers on the tube or using a vortex mixer. When using a vortex mixer, start the process slowly. Do not allow broth to get into the cap, lose control of the tube, or allow broth to come out of the tube

Disposal of Used Materials

Materials should be discarded in the appropriate areas for eventual autoclaving (steam sterilization) or incineration (burning).

mixed culture

A culture with many different species of bacteria

Isolation

the separation of different species in a sample or culture medium from each other

pure culture

Isolated bacteria can be placed in new culture medium to have only one species of bacteria

streak plate and pour plate

two common methods of isolation

streak plate method

a bacteriological loop is used to obtain a sample or culture medium using aseptic technique. The loop is then streaked across a nutrient agar surface in a specific manner. As the streaking progresses the bacterial cells are spread out (thinned out), farther and farther apart from each other. The separated cells reproduce and form a colony separated from other colonies. The colony consists of identical cells, which are called clones, because they are all descended from a single, original cell. If the original sample contained more than one type of bacteria, they should be isolated into separate single colonies

pour plate method

a loop is used similarly to sample material. Serial transfers are made to additional tubes so that each transfer results in a greater dilution of the bacteria present in the original medium or sample. If the dilution has been adequate, bacteria will be separated sufficiently in the solidified agar, so that individual colonies will form

morphology

bacteria in a variety of shapes

arrangements

association with each other

cocci

spherical bacteria

bacilli

Rod shaped bacteria

spirilla

spiral shaped bacteria

vibrios

slightly curved rods

coccobacilli

short rods

spirochetes

flexible spirals

pleophorphism

variety of shapes seen in a given sample

diplococcus

pair of cocci

diplobacillus

rod-shaped double bacillus

streptococcus

chain of cocci

streptobacillus

chain of rods

tetrad

a group of four cells

sarcina

cube shaped arrangement of cells

filamentous

thread-like and branching pattern of growth

negative stain

useful in observing morphology, size, and arrangement of bacterial cells

chomogen (nigrosine)

acidic (gives up a hydrogen ion) and carries a negative charge

smear prep

To view bacteria microscopically, the bacteria are applied and adhered to a microscope slide so they can be colored by chemical reagents called stains. The application of bacteria to a slide for the purpose of staining is called a smear. Smears can be made from bacteria growing in liquid and solid culture media.

Gram stain

crystal violet, iodine, alcohol, safranin

differential stain

uses various dyes to differentiate two large groups of bacteria based on their cell walls (peptidoglycan layer)