Antiseptics
Reduce the concentration of microbes on the skin
Steps to safely wash hands
- Wet your hands with clean running water and apply soap-use warm water if it is available
- Rub hands together to make a lather and scrub all surfaces
- Continuing rubbing hands for 20 seconds
- Rinse well under running water
When to wash your hands
Entering the lab
Leaving the lab
Any time in between
Media can be either a broth or a
Agar
Media can be either undefined or
Defined
Undefined media
The exact ingredients and their amounts are not known
Commercially available and easy to prepare
Defined media
All of the ingredients and their amounts are known
Preparation is more complicated and time consuming
Agar originates from
Seaweed
Agar is a better solidifier than gelatin because
It is firmer/stronger and cannot be degraded by bacteria
What is the most important thing when preparing media?
Sterility
The most effective way to sterilize media is by
Autoclaving
What are the three parameters critical in obtaining sterility?
Temperature
Time
Pressure (from steam)
Why are all three parameters necessary?
To kill endospores
How many ways can steam be released from the chamber of an autoclave?
2
When is fast exhaust used?
Dry items like glassware and certain plastics
When is slow exhaust used?
when working with liquids
Autoclave safety requirements are
Insulated gloves
Face shield
Slow opening of the door
Cart available to place hot items on
How can you tell whether something has been autoclave and is sterile?
Autoclave tape will turn striped
What kind of media is Tryptic Soy
Undefined
What parameters are used for autoclaving (specific)
20 minutes, 121C, 15 psi
What do aseptic techniques do?
Prevent media from becoming contaminated
What are some tools used in aseptic transfers?
Inoculating loops and needles
Sterile transfer pipette
Glass spreading stick
Describe the proper flame for a Bunsen burner
Inner and outer cone
What is the goal of isolation
To obtain a pure bacterial culture from a mixed culture
What happens when the last quadrant streak overlaps with the 1st quadrant streak?
Very few if any well isolated colonies
What happens when the loop is not flamed between quadrants?
No isolated colonies
What is ubiquity
Existing or being everywhere
What types of microorganisms are out there
Pathogenic
Opportunistic Pathogens
Free-Living Microorganisms
Why would you streak an environmental sample differently than a lab sample
Environmental sample will more likely have fewer organisms
Environmental Streak
One large zig zag across the entire plate
What are the forms a colony can take?
Circular
Irregular
Filamentous
Rhizoids
What ways are the elevation of a colony described
Raised Convex Flat Umbonate Crateriform
How are margins defined for colonies?
Entire Undulate Filiform Curled Locate
How can colonies be described?
Form Elevation Margin Pigmentation Texture Growth Patterns
When making a dilution what are important points to remember
Always add the sample to the saline broth
Always mix each dilution tube after adding the sample
Always change piper tips after each sample transfer
Where should you place the bacterial dilution in the agar plate?
The center
Colony forming units
Can be calculated when you know the number of colonies at a specific dilution. CFU/mL =#colonies/(dilution factor *volume plated)
Biofilm
A complex association that arised from a mixture of microorganisms growing together on the surface of a habitat
Stages of biofilm formation
- Surface conditioning
- Initial attachment
- Slime formation
- Secondary colonization
- Maturation
- Dispersal
Are bacteria within a biofilm more or less resistant to disinfectants/detergents as well as antibiotics
More
What is the media enriched with to help with biofilm formation
Glucose to help with surface conditioning
What dye was used to observe the biofilm
Crystal violet
What is soil composed of?
40% inorganic material
5% organic material
50% Air and water
5% microorganisms and macroorganism
Decomposed plant material lays
Zero to 2 inches into the soil
Surface soil
Soil that is 2 to 10 inches and is high in organic matter and microbes
Subsoil
10 to 30 inches little organic matter and fewer microbes
Soil base
30 to 48 inches with even fewer microbes
What roles can microbes play
Carbon cycle - detritivores
Nitrogen cycle-denitrifying bacteria, nitrogen fixing root bacteria
Sulfur cycle
Food web
Prokaryotes in Soil
Bacteria
Eukaryotes in Soil
Filamentous fungi
Protozoan
Nematode
What types of bacteria are in soil?
Chemoautotrophs Decomposers Mutualists Disease suppressors Pathogens
What do chemoautotrophic bacteria do
Oxidize inorganic compounds to yield energy and reduce CO2 Ex. Nitrosamines europaea Nitro so after winogradskyi Thiobacillus Desulfovibrio
What do bacterial decomposed do?
Break down or gain biomass Ex Pseudomonas fluorescens Bacillus subtitles Actinobacteria
Many members of the actinobacteria group produce?
Antibiotics
What are bacterial mutualists?
Bacteria that form partnerships with plants
Ex
Rhizobium
What re disease suppressors
Bacterial species that can suppress the occurrence of plant disease by secreting anti-fungal or anti-insecticidal compounds Ex Bacillus Rhizoctonia Bacillus thuringiensis
What are bacterial pathogens
Bacterial species that can cause disease in plants and/or animals
Ex
Erwinia
Clostridium tetani
What types of eukaryotes are in soil
Decomposers
Mutualists
Pathogens
What are fungal decomposers
Species that break down organic biomass
Ex
Aspergillus
Penicillium
What are fungal mutualists
Bacteria that form partnerships with plants
Ex
Mycorrhizal fungi
What are fungal pathogens
Fungal species that can cause disease in plants and/or animals
Ex
Verticillium
Nutrient agar promotes the grow of ______ but not _____
Many types of bacteria; fungi
Glycerol yeast extract supports the growth of _____ but not enough to support_______ or _______
Actinobacteria; simple bacteria; fungi
Sabouraud Dextrose Agar with antibiotics will allow _____to grow but not ______
Fungi, bacteria
What kind of viruses cannot be worked on in this lab?
Animal
Working with animal viruses requires a high level of sterility
Work must be done in biosafety cabinets that have a filtered air supply
What is a bacteriophage?
Viruses that only infect bacteria
Bacteriophage require a host _______ in which to replicate because they do not have _______,_________ etc, and use the cellular_______ of its host. They contain ______ and are ________ in structure.
Bacteria; enzymes; ribosomes; machinery; DNA; complex
Animal viruses require a host _____cell and use the cellular________of its host. They contain _____or_____but never both and are _______in structure.
Eukaryotic; machinery;DNA;RNA;simple
A bacteria phage consists of
Caps is or head with dna inside
Sheath
Tail fibers
Animal viruses contain
Capsid with DNA
Phases of bacteriophage infection
Attachment Penetration Synthesis Assembly Release
Why is soft agar used for plaque assays?
Bacteria cannot move through it but bacteriophage can move short distances
What is a plaque
The area of clearing on the bacterial lawn.
How do you calculate PFU/mL
of plaque/(dilution factor * ml of Phage added to e. Coil)
Can different phage produce different looking plaques?
Yes
How many sets of lenses does a compound microscope have?
2
How many hands should be used when carrying a microscope?
2
Before you turn on the microscope check the ____ ______.
Lamp control
What 4 objectives are there?
4x
10x
40x
100x
What is the magnification of the ocular lens?
10x
What is a condensor?
The piece that narrows the beam of light that strikes the specimen
What must be used when viewing specimen under the 100x objective?
Immersion oil
Why must immersion oil be used with the 100x objective lens?
It prevents the light from being refracted.
List types of microorganisms
Viruses. Helminths Bacteria Archaea Fungi Algae Protozoa
Viruses can only be seen using a ______microscope
Electron
Most bacteria and archaea can be seen by a light microscope using ______ ________ and the aid of ________.
Oil immersion; staining
Bacteria that do not require staining
Cyanobacteria because of photosynthetic pigments Ex Gloeocapsa Anabaena Oscillatoria
Aquatic fungi are from the class
Chytridiomycetes
Types of algae in freshwater
Chlorella Ulothrix Spirogyra Diatoms or phytoplankton Navicula Synedra Cocconeis
Protozoa include
Euglenids - green flagellated photosynthetic Protozoa
Dinoflagellates - flagellated Protozoa
Ciliates - use cilia for locomotion
Sarcodina- use pseudopods for locomotion
Helminths include
Nematodes-unsegmented with a full digestive tract
When using a slide cover which objective should you not use?
100X
Fungi can be ______ or _______
Macroscopic; microscopic
Microscopic fungi have two basic morphological types which are
Yeasts and Molds
General characteristics of yeasts
Grow in colonies that are very similar in appearance to bacterial colonies
Yeast cells are round or oval in shape
Reproduce by budding
Example of yeast
Candida albicans - oral thrush - opportunistic pathogen
General characteristics of molds
Grow as hyphae (long thread-like cells)
Hyphae can be septate(divided by cell walls) or nonseptate(a continuous cell) and vegetative or reproductive.
Primary mode of reproduction is by asexual spore formation
Asexual spore types Nicole sporangiospores (enclosed) and condidospores (free)
Sexual spore formation is some molds involves production of zygospores
Examples of mold
Rhizopus- includes common bread mold-sporangium, nonseptate, form zygospore
Aspergillus - opportunistic pathogen, septate, conidiophores
Penicillium - produces penicillin, septate, conidiophores
Why do stains need to be used to view most bacteria?
Cytoplasm is transparent
What is a stain?
A solution in which a dye or chromogen has been added to a liquid
A chromogen consists of __ components. The _____ and the _____.
2; chromosphore; auxochrome
The _____ is carried by the auxochrome and determines whether a or not a stain will adhere to a bacterium
Charge
Can be positive or negative depending on the charge carried by the auxochrome
Bacteria have a ______charge on their cell surface
Negative
A Positive stain has a _______charge so the _______ binds to the bacterial cell surface.
Positive; auxochrome
The bacteria is stained & background is white
With a negative stain, the auxochrome has a ________ charge which ______ and the _________ cannot bind to the bacterial cell surface.
Negative; repel; auxochrome
Bacteria left colorless and background is stained.
Examples of Positive Stains
Crystal violet
Methylene blue
Safranin
Examples of negative stains
India Ink
Nigrosin
Positive stains require ____ fixing; negative stains do not.
Heat
What are the pros to simple and negative stains?
Only require 1 dye (uncomplicated)
What are the cons of simple and negative stains
They cannot differentiate unique cell structures or features between bacteria.
All negative stained slides must be _______ in the ______ _______.
Discarded; bleach bucket
What are the three general shapes of bacteria?
Bacillus (rods)
Coccus (spheres)
Spirillus (spiral)
What are some types of arrangement?
Singles or pairs
Chains
Clusters of cocci
Palisades of bacilli
What is pleomorphism
Slight deviations in the overall shape and/or size of a bacteria
Who invented gram staining?
Christian Gram
What makes the gram stain so important?
It can differentiate between bacteria
Gram ______ cells have a thick layer of peptidoglycan; Gram ______ cells have a thin layer of peptidoglycan and an outer membrane.
Positive; Negative
Gram staining requires ________ fixation
Heat
What is the primary stain in gram staining?
Crystal violet
The mordant used is _______ and produces _______-_________ complexes
Iodine;crystal violet;iodine
What decolorizer is used?
Ethanol
How does the decolorizer work?
Dissolves the lipids in the outer membrane
What counterstain is used?
Safranin
What bacteria does gram staining not work on?
Nocardia and Mycobacterium
What color is a gram positive bacteria?
Purple
What color is an acid fast stain positive?
Pink
What do acid fast positive bacteria have in their cell walls?
Mycolic acid (waxy lipid)
Example of a Mycobacterium
Mycobacterium tuberculosis
Highly communicable
1/3 of the world is infected with it
Leading killer of hiv infected individuals
4.2/100000 people in the US
Can acid-fast non-pathogenic mycobacterium and nocardia species exist in the environment?
Yes
what is the primary stain in acid fast staining?
Carbolfuchsin
Phenol based that is lipid soluble
Carbolfuchsin stain can be enhanced by what two methods?
Applying heat to drive the steam in (steam)
Increase the concentration of phenol
If heat is used in the staining procedure, which method is it?
Ziehl-neelsen
If a higher concentration of phenol is used in the carbolfuchsin stain which method is it?
Kinyoun Method
What is a capsule?
A complex layer of sugars and proteins that tightly surrounds a bacterium
Capsule Staining is a _______stain.
Differential
What is the role of the capsule?
Protection against environmental conditions and dehydration
Adherence to surfaces
Prevents phagocytosis
In a pathogenic bacterial species a capsule is considered a ______ ______.
Virulence factor
Can non pathogenic bacteria possess capsules?
Yes
Examples of capsule bacteria
Neisseria menigitidis
Many bacillus species including those found in the soil bacillus megaterium, bacillus licheniformis
Can a capsule be stained?
No
Why wont a capsule stain?
It is water soluble
The capsule stain is a differential stain that combines ______ and _____ stains.
Negative; simple
Congo red is an ______ dye (ie. it carries a ________charge)
Acidic; negative
In a negative stain only the ______ will be stained
Background
A capsule stain slide is never _____ _______.
Heat fixed.
What is the simple stain used in capsule staining?
Maneval’s stain
Maneval’s stain is a ______ dye with a _____ charge.
Basic; positive
Capsule stain slides must be discarded in the ______ _______
Bleach; bucket
To overcome this constant flux of nutrients in the environment, some bacteria have the capacity to produce _____ ______ or ________.
Inclusion bodies; granules
Poly B-hyroxybutyrate (PHB) is an _____ _____ _____
Inclusion body stain
If a bacterium is in a nutrient rich environment, it is likely to contain ______ or ______ inclusions
More; larger
Why produce inclusion bodies?
Nutrient reservoir
Avoid lyse from an increase in osmotic pressure
Do all bacteria produce inclusion bodies?
No
Carboxysomes are an ________ that continue the enzyme _______.
Inclusion; ribulose 1,5-diphosphate carboxylase
Types of inclusions:
Suffer Granules (Thiobacillus)
Carboxysomes
Polysaccharide Granules - contain glycogen/starch
Metachromatic granules - volutin (inorganic phosphates) for ATP
Corynebacterium diphtheria is a metachromatic granules that causes diphtheria
Magnetosomes contain iron oxide which can act like tiny magnets
Lipid inclusions contain PHB poly B-hyroxybutyrate used as both carbon and energy sources
PHB consists of
B-hyroxybutyrate molecules linked by an ester bond between carboxyl group of one an the hydroxyl group of the next
Hydrolysis of the ester bound results in carbon containing molecules and sufficient free energy to be harnessed for atp
PHB Inclusions range in diameter from ____ to ____
0.2; 0.7
PHB inclusions are contained within a unique membrane that is composed of a _______ lipid layer
Single
Can all bacteria contain PHB inclusion?
No
What is the primary stain of PHB?
Sudan Black B
Sudan Black B is a ______ soluble stain that is ______
Lipid; neutral
The PHB counterstain is
Safranin
Certain bacteria can survive in extremely unfavorable conditions by forming _______
Endospores
Endospores is able to withstand extreme ______, ________, ________, and _______ ________.
Temperature; disinfectants; desiccation; and UV radiation
Major characteristics of endospores
Contain dna but are inactive metabolically (dormant)
Consist of a very tough outer cell coat, a cortex, and an inner core (3 C’s)
What are the 3 C’s
Coat, cortex, Core
Only a few species can form endospores; two of which are _______ and _______
Bacillus; Clostridium
Mostly non pathogenic however bacillus antracis causes anthrax in humans and clostridium tetani cause tetanus and clostridium botulinum cause botulism.
The _____, ______, and ________ of the endospores within a bacterium can help in the identification of the organism.
Size; shape; location
What is the primary dye in endospores staining?
Malachite green
Does endospores staining use steam to drive the stain into the bacteria?
Yes
What is the counter stain in endospore staining?
Safranin
Flagella may contribute to the _______ of a bacterium since it can allow the bacterium to move to its preferred anatomical site to start the disease process.
Virulence
What are two ways to determine whether a bacterium is motile?
Hanging drop slide
Semi-solid motility agar
The different arrangements of flagella
Monotrichous = 1 flagella at 1 pole Lophotrichous = several flagella at 1 pole Amphitrichous = 1 flagella at each pole Peritrichous = flagella all over
What is used to thicken the flagella so that it can be seen?
Mordant
Flagella are thin and ________
Delicate
The mordant is _____ meaning it will stick to the flagella as well as any dust or dirt on the slide
Non-specific
What is motility media
A semi solid media which has just enough agar added to produce a media with the consistency of sloppy jello (4x less agar)
Motility media is thick enough to allow motile bacteria to move but too thick for __________ bacteria to disperse
Non-motile
What is added to the motility media to make a motility test easy to read?
Tetrazolium salt
TTC reduces to formazan and ______
HCL
Formazan is _____ and ______
Red; insoluble
TTC is reduced by _______ ______ bacteria
Metabolically active