Microbiology Labs

Tryptic Soy Agar (TSA)

support the growth of bacteria and fungi

Clamshell Technique

Using the lid of the petri dish plate to shield the media from airborne contamination

Inverted Plate Incubation

avoiding condensation dripping onto media and causing contamination

Culture

Media that contains living microbes

Pure culture

culture that contains a single species, well isolated colony

Aspectically

without contamination of yourself, others, the environment and the source culture

Broths

used to grow microbes when fresh cultures or large numbers of cells are required

Agar slants

used to grow stock cultures that can be refrigerated after incubation for several weeks

Plated media

used for obtaining isolation of species, differential testing and quantifying bacterial densities

Sabouraud Dextrose Agar (SDA)

a selective media used to grow fungi

Recipe for SDA

• Penicillin and Streptomycin

• pH 5.2-5.6

Colony

Visible mass of microorganisms all orginating from a single mother cell

Colony Elevation

Convex

Flat

Umbonate

Pulvinate

Really Pulvinate

Colony Margin

Lobate

Undulate

Smooth/Entire

Rhizoid

Filamentous

Streak Plate Method

Used to isolate individual colonies of bacteria from a mixed culture

Colony Surface

Smooth

Moist

Mucoid

Rough

Wrinkled

Dry, powdery

Influencing bacterial growth

Incubation time and temperature

Rhizopus-mold

Aspergillus-mold

Penicillium-mold

Smear prep

Bacterial emulsion thinly spread out into a smear on a glass slide and allowed to dry

Staining techniques

• Simple stain (single dye)

• Negative stain (single dye)

• Capsule stain (2 dyes or counterstain)

• Spore stain (2 dyes or counterstain)

• Acid-Fast stain (2 dyes and decolorization)

• Gram stain (2 dyes and decolorization)

Simple stain

uses alkaline dye that stains all cells the smae color

Possible Dyes for Simple Stain

Crystal Violet, Methylene Blue, Safranin

Negative Stain

Uses acidic dye that repells negatively charged bacterial surface

Possible dyes for Negative Stain

India Ink, Congo Red, Nigrosin, Eosin

Endospores

highly resistant to heat and chemicals, which allows them to survive in environmental conditions unsuitable for growth

Auxochrome

charged portion of a chromogen and allows it to act as a dye through ionic or covalent bonds

Chromogen

colorless compound that can be converted into a colored dye or pigment

Gram Stain

Used to distinguish between gram-positive and gram-negative cells as well as determining cell morphology, size and arrangement

Mycolic acid

a waxy substance that gives acid-fast cells a higher affinity for the primary stain and resistance to decolorization by an acid alcohol solution

Acid-Fast Staining Procedures (2)

Ziehl-Neelsen (ZN) method - heat used

Kinyoun (K) method - cold stain

Media Types

Broth - Liquid (0%)

Deep - Semisolid (~0.4%)

Slant or Plate - Solid (~1.5%)

Turbidity

Relative population size

Selective medium

contains ingredients to inhibit growth of certain species and/or allow the growth of other species

Differential medium

contains specific ingredients or aspects to indicate if a species that possess or lacks a biochemical process

7.5% NaCl Selectivity

Selective for Staphylococcus species b/c they are osmotolerant

Mannitol and Phenol Red Differential

Differential b/c pathogenic Staphylococcus species ferment the sugar mannitol (produce acid)

Cardinal Temperatures

Psychrophile

Mesophile

Thermophile

Lawn

looks like a lawn, covered all over and with bacterial colonies merging with each other

Acidophiles

grow at extreme acid pH, below 5.5

Neutrophiles

Majority of organisms grow at pH between 5.5 and 8.5

Alkalinophiles

grow at extreme alkaline pH above 8.5

Normal Saline (NS)

0.91% of NaCl

Extreme halophiles

require salt conc. of 15-30%

Halophiles

require a high concentration of salt (hypertonic) >3% up to 15%

Osmotolerant (halotolerant)

do not require high concentration of solute but can tolerate it when it occurs

Capsule Stain

a differential stain used to detect cells capable of producing an extracellular capsule

Capsules

composed of mucoid polysaccharides or polypeptides that repel most stains because of their neutral charge

3 genera with ability of Endospores

Bacillus, Clostridium and Sporosarcina

Acid Fast Stain

used to detect cells capable of retaining a primary stain when treated with an acid alcohol

Gram Positive and Negative Colors

Positive- Purple

Negative- Pink

Gelatinase Test Results

Negative- Solid

Positive - Liquid

Melts @ 28C

Gelatinase Test

Used to determine the ability of a microbe to produce gelatinases

Crateiform liquefaction

form of liquefaction where the microbe is unable to completely breakdown gelatin into its component amino acids

Basic reactions of litmus milk

• Lactose Fermentation

• Reduction of litmus

• Casein coagulation

• Casein hydrolysis

Litmus milk test

differentiate members within with genus Clostridium, differentiates Enterobacteriaceae from other gram negative bacilli

Motility Test

detect motility as diffuse growth radiating from the central stab line

SIM medium

used for determination of 3 activities: sulfur reduction, indole production, indole production, motility

Sulfur reduction to H2S

Cysteine desulfurase

Thiosulfate reuctase

Tryptophan hydrolysis (SIM medium)

addition of Kovac’s reagent after incubation (presence of tryptophanase)

Species Name: Clonorchis sinensis

Phylum: Platyhelminthes

Class: Trematoda

Species Name: Schistosoma mansoni

Phylum: Platyhelminthes

Class: Trematoda

Species Name: Schistosoma mansoni- cercaria larva

Phylum: Platyhelminthes

Class: trematoda

Species name: Taenia pisiformis or Taenia solium - scolex

Phylum: Platyhelminthes

Class: Cestoda

Species name: Taenia pisiformis or Taenia solium - immature proglottid

Phylum: Platyhelminthes

Class: Cestoda

Species Name: Taenia pisiformis or Taenia solium - mature proglottid

Phylum: Platyhelminthes

Class: Cestoda

Species name: Taenia pisiformis or Taenia solium - gravid proglottid

Phylum: Platyhelminthes

Class: Cestoda

Species Name: Dipylidium caninum- scolex

Phylum: Platyhelminthes

Class: Cestoda

Species name: Dipylidium caninum - immature proglottid

Phylum: Platyhelminthes

Class: Cestoda

Species name: Dipylidium caninum - mature proglottid

Phylum: Platyhelminthes

Class: Cestoda

Species name: Dipylidium caninum - gravid proglottid

Phylum: Platyhelminthes

Class: Cestoda

Species name: Ascaris lumbricoides

Phylum: Nematoda

Species name: Wuchereria bancrofti

Phylum: Nematoda

Oxidation-Fermentation (O-F) Test

identify whether an organism can oxidize carbohydrates and/or ferment carbohydrates

Citrate Test

the ability of organisms to use citrate as their sole carbon source and perform citrate fermentation

Citrate (+) organisms

make the enzyme citrase or citrate-permease to transport the citrate into cell

MR Test

detect organisms capable of performing a mixed acid fermentation, which overcomes phosphate buffer in the medium and lowers pH

Voges-Proskauer Test

designed for organisms that are able to ferment glucose, but quickly convert their acid products to acetoin and 2,3-butanediol

Ultraviolet light application

used to disinfect laboratory and health-care environment work surfaces and surrounding air

Sterilization

a process that destroys all viable microbes, including viruses and endospores

Disinfection

process to destroy vegetative pathogens, not endospores

Bacitracin

peptide antibiotic that inhibits bacterial cell wall synthesis, only effective on bacteria that have cell walls and in process of growing

Novobiocin

antibiotic that interferes with ATPase activity during DNA replication

Optochin

antibiotic that interferes with ATP synthase activity and ATP production

Bacterial resistant

Antimicrobial is ineffective against bacteria

Bacterial intermediate

antimicrobial might be effective against bacteria

Bacterial Susceptible or Sensitive

Antimicrobial effectively kills or inhibits bacteria (appropriate to treat infection)

Zone of Inhibition (ZOI)

Determines whether a bacterial species is resistant, intermediate or susceptible to an antimicrobial

Kirby-Bauer Test

Disk diffusion test to measure the effectiveness of antibiotics and other chemotherapeutic agents on pathogenic microorganisms,

Minimum Inhibitory Concentration (MIC)

lowest concentration of an antimicrobial drug that prevents visible growth