Practical Flashcards
Selective:
designed to suppress the growth of some microorganisms while allowing the growth
of others.
• Example: Mannitol Salts Agar (high salt concentration), MacConkey Agar (crystal violet)
Differential:
differentiates between organisms through an indicator within the medium that
distinguish different chemical reactions
• Example: Mannitol Salts Agar (mannitol fermentation), Blood agar (ability to break
down RBC’s)
Complex/Undefined media
Exact composition of ingredients in the broth/agar is unknown TSA/TSB o Beef extract 3 grams o Peptone 5 grams o Distilled water 1,000 mL
Synthetic/Defined media
Exact quantities of all ingredients are known NH4H2PO4 1 gram o Glucose 5 grams o Sodium chloride 5 grams o MgSO4 x 7H2O 0.2 gram o K2HPO4 1 gram o Distilled water 1,000 liter
Methods of Sterilization
Autoclaving Dry Heat Sterilization Filtration UV radiation Chemical sterilization
Autoclaving
o 121° C of steam, 15 psi, 15-20 minutes or longer depending on volume
o For liquids that are not heat sensitive
Dry Heat Sterilization
o 160-170° C for 2 hours
o For glassware
Filtration
o For heat sensitive material
UV radiation
o For surface sterilization
Chemical sterilization
o For heat sensitive materials
o Ethylene oxide – used often in hospital settings
Storage of media
Short-term storage
Long-term storage
- Capping with a cotton plug, plastic, or foam cup
* Capping with metal or screw caps
Culture Maintenance: Short periods (1-2 months) where cultures are used frequently Long periods of storage for cultures not used frequently
- Refrigeration
* Desiccation drying or Lyophilisation freeze drying
A colony
has cells that are genetically identical because they arise from a single cell.
mixed culture
a culture that contains more than one type of organism
pure culture
a culture consisting of one type of organism
Colony forming unit (CFU
measure of viable bacterial cells within a sample per milliliter
Limit of Resolution (or resolving power)
actual measurement of how far apart two points must be for the microscope to view them as being separate
Best limit of resolution 2um
numerical aperture
a measure of a lens’s ability to capture light coming from the
specimen and use it to make an image.
Parfocal Lens System
An optical system that stays fairly in focus when magnification on the
objective lens is changed
Three domains
Bacteria, Archaea, Eukarya
Prokaryotes vs. Eukaryotes
- Prokaryotes: 16S rRNA
* Eukaryotes: 18S rRNA
Spirillum volutans
spiral shaped
Mycobacterium tuberculosis
acid fast bacterium
Klebsiella pneumoniae
capsule stain bacterium
Proteus vulgaris
contains flagella (peritrichous arrangement)
Cyanobacteria:
Nostoc
contains heterocysts for N2 fixation
Protozoa:
Euglena
MOtility by lfagella and uses longitudinal fission to divide
Subphylum Sarcodina-amoebas
Amoeba proteus
Movement by pseudopods via cytoplasmic streaming and divides by binary fission
Subphylum Sarcodina- ameobas
Entamoeba histolytica
Causitive agent of amoebic dysentery
Phylum Ciliophora-ciliates
Paramecium
motile by cilia and uses transverse fission to divide
Phylum Apicomplexa
sporozoans
Phylum Kinetopplastida
Leishmania donovani
Parasite that causes leishmaniasis
Transmitted through the bite of a sand fly
Open sores on skin that form after weeks or months oftransmission.
Photosynthestic protists
Spirogyra
Green, has filamentous algae and chlorophylls A&B
Photo sythetic Protists
Volvox
Flagellated algae
Fungi
nonmotile, absorptive heterotrophs, saprophytes, and are parasitic to plants, animals, and humans sometimes.
Yeast- unicellular
Saccharomyces cerevisiae
ascomycete used in food industry
Yeast-unicellular
Candida albicans
Part of normal flora
Yeast-unicellular
Cryptococcus neoformans
a casual agent of pneumonia and meningitis
Molds-filamentous
Hyphae are individual fungal fragments
mycellium are a collection of hyphae
Rhizopus
Zygomycete black bread mold
Molds-filamentous Hyphae are individual fungal fragments mycellium are a collection of hyphae Aspergillus Penicillin
Deuteromycete
Deuteromycete
Importance of Heat fixing
- Kills the cells so that stains penetrate better
- Proteins are denatured so the cells stick better to the slide
- The slide can be kept longer
Simple Staining
A single stain/dye is used to create a contrast between the cell and the background. Simple
staining is used to see the shape, arrangement, and size of the microbe.
Basic Dyes
Positively charged so that it attaches to the negatively charged bacterial membrane Example: crystal violet, carbolfuchsin, methylene blue
Acidic Dyes
Negatively charged so that it is repelled from the bacterial membrane and stain
background (negative staining)
Example: nigrosin
Differential staining
stains whole cells
• Gram stain
• Acid Fast stain
Structural stain
stains parts of cell
- Endospore stain
- Flagella stain
- Capsule stain
What is a Gram variable result?
After 24 hours, the thick peptidoglycan layer of the cell wall becomes leaky and the crystal violet-iodine complex will not set upon the alcohol rinse.
3% KOH test
An alternative to the gram stain. Bacterial colony mixed with 3% KOH. Within 60 seconds it is indicative of a gram negative culture is viscous and stringy. Gram negatice cells lyse and release cellular DNA.
Endospore
A dormant and highly resistant form of bacterium that can survive in harsh and stressful conditions that would normally kill it to preserve its genetic material. Keratin is apart of their outer covering that helps with stand tough environment.
Sporulation
When harsh conditions arise, the bacteria will cease normal growth and produces the resistant endospore.
Vegetative cells
Cells without an endospore
Sporangium
The part of the cell surrounding the mature endospore
Central spores
Spores located in the middle of the sporangium
Terminal spores
Spores at the end of the sporangium
Free spores
Spores released into the environment
Endospore stain
Differential stain used to detect the presence and location of spores in bacterial cells.
Genera that produce spores
Bacillus,
Clostridium,
Sporosarcina,
and Sporolactobacillus
Bacillus anthracis
anthrax
Clostridium tetani
tetanus
Clostridium botulinum
botulism
Clostridium perfringens
gas gangrene
Clostridium diffcile
pseudomembranous colitis
Endospore can be cultivated in lab using
NSM Nutrient Sporulation Media
contains calcium, maganese, and maganesium to promote sporulation.
Negative Stains
Used for bacteria who are to delicate for heat fixing. Negative stains contain chromogens that have negative charge that repel off the negative membrane. India ink nigrosin, congo red, and eosin are negative stains.
Klebsiella pneumoniae
The casual agent of pneumonia
Acid-Fast Stains
Is a differential stain for bacteria with mycolic acis in their cell walls.
Ziehl-Neelsen method
Used to stain acid-fast bacteria
Carbolfuchsin
Primary stain in acid-fast and contains phenol. Helps dissolve some of the liped on the waxy cell wall.
Acid alcohol
Removes carbolfuchsin on negative acid-fast bacteria.
Methylene blue
Counterstain that will stain acid-fast negative cells
Mycobaterium leprae
acid-fast leprosy
Mycobacterium tuberculosis
acid-fast tuberculosis
Nocardia
acid-fast
Cryptosporidium oocysts
acid-fast causes diarrhea in humans
Isopora oocysts
acid-fast
Capsule stain
Starts as a negative stain because the capsule is not stained but the background is. Once the background is stained the cell within the capsule is stained with crystal violet or safrinin
Capsule
made up of mucoid polysaccharides or peptides and is heat sensitive
Virulence
the degree to which a pathogen can cause a disease
Bacillus anthracis
Contains a capsule
Steptococcus pneumoniae
Contains a capsule
Flagella Stains
Mordants are used to increase thickness of the flagella. Then the flagella are stained with a dye such as basic fuschin.
Flagella
Are longthreadlike appendages which provide motility
Flagella arrangments: monotrichous Lophotrichous Amphitrichous Peritrichous
- Single flagellum (polar)
- Tufts of flagella at the end of the cell
- Single flagellum at each end of the cell
- Flagella emerging from the entire cell surface
Proteus vulgaris
May cause UTI’s, bacteremia, and pneumonia
Brownian movement
Is when a bacterial cell is stationary and exhibits a shaking motion due to the bombardment of water molecules.
Aerotolerance
The ability or the inability to survive in the presence of oxygen
Obligate aerobes
Microbes will only grow in the presence of oxygen
Obligate anaerobes
Microbes will grow in the absence of oxygen
Facultative
Microbes grow in the presence or absence of oxygen
Microaerophiles
Microbes that grow below the surface of the medium because they require a lower than atmospheric oxygen tension for normal growth
Gas pak anaerobic system
packet contains inorganic carbonate, activated carbon, ascorbic acis, and water. inorganic carbonate converts into carbon dioxide
Catalase Test
Used to differentiate between similar groups of bacteria such as Staphylococcus and micrococcus which are positive, versus Lactococcus and Enterococcus which are catalase negative.
-Hyrogen peroxide will bubble if positive
Flavoprotein
Catalase test
One carrier molecule of electrons to oxygen used in the ETC
When flavoproteins carry electrons down the ETC, two cellular toxins, ___ and ____ are formed
Hydrogen peroxide and superoxide molecule O2-
Superoxide dismutase
an enzyme that catalyzes the destruction of the superoxide molecule into hydrogen peroxide and oxygen
Catalase
An enzyme by some obligate aerobes and facultative anaerobes that break down the hydrogen peroxide into water and atmospheric oxygen
Oxidase Test
Used in clinical procedure. Detects the presence of cytochrome C.
Cytochrome C
Carrier molecule that transfers electrons to oxygen in the electron transport chain.
Cytochrome C Oxidase
An enzyme found in some obligate aerobes, facultative anaerobes, and microphiles that catalyze the transfer reaction. Cytochrome C becomes oxidized while O2 becomes reduced.
Oxidase positive
Neisseria (gonorrhea) and Vibrio (cholera) blue strip color change
Oxidase negative
Enterobacteriaceae (enteric bacteria)
Nitrogen Reduction Test
It tests the ability of an organism to reduce nitrite to nitric oxide, or molecular nitrogen.
Positive Nitrate Reduction Test
medium turns red after reagent A and reagent B has been added OR no color change even after zinc has been added.
Negative Nitrate Reduction Test
Medium turns red after the addition of zinc
Nitrate reductase
Used in nitrate broth to reduce nitrate to nitrite
Nitrate Reduction Test Reagent A
Sulanilic acid
Nitrogen Reduction Test Reagent B
Alpha naphthylamine
Mannitol Salt Agar
Selective (7.5% NaCl) and differential (mannitol and phenol red) medium.Bacteria that can survive the high salt content flourish on this media. MSA isolates pathgenic bacteria from non-pathogenic
Mannitol Salt Agar pH indicator
Phenol Red pH: 6.8 or below turns yellow (fermentation of acid) 7.4-8.4 stays red 8.4 and above media turns pink
MacConkey Agar
Selective and differential medium. Bile salts and crystal violet make it selective by inhibiting growth of Gram-positive bacteria. Neutral red and lactose makes it differential. Acid is formed when certain species of enteric bacteria ferment lactose
MacConkey Agar pH indicator
Neutral red.
pH:
6.8 and below lactose fermentation colonies are red
6.8 and above lactose not fermented Growth of the colonies colorless or transparent
Salmonella typhi & Shigella dysenteriae
Do not ferment lactose
Escherichia coli, Enterobacter, and Klebsiella
Lactose fermenter
