MicroLab - Long quiz 1

midterm topics

biological safety levels

• series of protections relegated to autoclave-related activities that take place in particular biological labs

• individual safeguards designed to protect laboratory personnel and the surrounding environment and community. 

• they are important because they dictate the type of work practices that are allowed to take place in a lab setting

• they heavily influence the overall design of the facility in question

centers for disease control and prevention (CDC)

• sets BSL lab levels as a way of exhibiting specific controls for the containment of microbes and biological agents

the lab levels are determined by the following

• risk related to containment

• severity of infection

• transmissibility

• nature of work conducted

• origin of the microbe

• agent in question

• route of exposure

biological safety level 1 (BSL-1)

• lowest of the four 

• personnel work with low-risk microbes that pose little to no threat of infection in healthy adults

• research taking place on benches without the use of special containment equipment

• not required to be isolated from surrounding facilities 

• example: 

  • non-pathogenic strains of E.coli 

standard microbial practices of BSL-1

• mechanical pipetting only (no mouth pipetting allowed) 

• safe sharps handling

• avoidance of splashes or aerosols

• daily decontamination of all work surfaces 

• hand washing

• prohibition of food, drink, and smoking materials

• personal protective equipment: 

  • eye protection

  • gloves

  • lab coat/gown

• biohazard sign

• immediate decontamination after spills, infectious materials are also decontaminated prior to disposal (autoclave)

biological safety level 2 (BSL-2)

• maintain the same standard microbial practices as BSL-1 labs

• includes enhanced measures due to the potential risk of the aforementioned microbes

• greater care to prevent injuries such as cuts and other breaches of the skin

practices required in a BSL-2 lab setting

• appropriate personal protective equipment (PPE) must be worn

• all procedures that can cause infection from aerosols or splashes are performed within a biological safety cabinet (BSC)

• an autoclave or an alternate method of decontamination is available 

• the laboratory has a self-closing, lockable doors

• a sink and eyewash station should be available

• biohazard warning signs

• access to the lab is more restrictive than BSL-1 lab

biological safety level 3 (BSL-3)

• includes work on microbes that are either indigenous or exotic, can cause serious or potentially lethal disease through inhalation 

• work is often strictly controlled and registered with the appropriate government agencies

• laboratory personnel are also under medical surveillance and could receive immunization for microbes they work with

• examples: 

  • yellow fever

  • west nile virus

  • bacteria that causes mycobacterium tuberculosis

  • bacteria: 

    • yersinia pestis

    • brucella abortus

    • chlamydia psittaci

    • pseudomonas mallei

  • viruses:

    • west nile fever

    • herpes B

    • hepatitis A

common requirements in a BSL-3 laboratory

• standard personal protective equipment must be worn, respirators might be required

• solid-front wraparound gowns, scrub suits, or coveralls are often required

• all work with microbes must be performed within an appropriate BSC

• access hand-free sink and eyewash stations near the exit

• sustained directional airflow to draw air into the laboratory from clean areas towards potentially contaminated areas

• a self-closing set of locking doors with access away from general building corridors 

• access to BSL-3 labs must be restricted and controlled at all times

biological safety level 4 (BSL-4)

• rare

• highest level of biological safety

• consist of work with highly dangerous and exotic microbes

• infections caused by these types of microbes are frequently fatal, and come without treatment or vaccines

• laboratory is extremely isolated - located in a separate building or an isolated and restricted zone of the building

• examples: 

  • ebola

  • marburg viruses

BSL-4 laboratories have the following containment requirements

• personnel are required to change clothing before entering, shower upon exit

• decontamination of all materials before exiting 

• personnel must wear appropriate personal protective equipment from prior BSL levels, as well as a full body, air-supplied, positive pressure suit

• a class-III biological safety cabinet

isolation of bacteria

• A primary method used to separate different groups of microorganisms.

• A technique that distinguishes groups of bacteria based on their growth patterns.

• Bacteria grow differently on various nutrient media depending on temperature, pH, and oxygen availability.

• Isolation of bacteria is essential for their identification and classification.

• The isolation process involves specimen collection, preservation, culturing, and microscopic examination.

• Specimens can come from clinical, environmental, or food samples.

• Specimens must be preserved under sterile conditions and transported quickly to maintain bacterial viability.

• Both culture and non-culture methods are used for bacterial isolation.

• In culture methods, bacterial growth is indicated by turbidity or colony formation in liquid or solid media.

• Non-culture methods like PCR and LCR are used to detect and identify bacteria.

• Microscopic examination after culturing and staining helps identify bacteria by color, shape, size, and other features.

• Bacterial isolation techniques help define and differentiate various bacteria

methods of isolation of bacteria

• pouring method

• spreading method

• streaking method

• serial dilution method

pouring method

• simplest method

• a bacteria suspension laden with a huge bacterial population is generally taken

procedure for pouring method

• Take 1 mL of the bacterial sample and place it into a sterile Petri dish.

• Bacterial growth requires a nutrient source such as carbon or nitrogen.

• Prepare and pour nutrient agar medium into Petri plates containing the bacterial sample.

• Rotate the plates clockwise and counterclockwise to evenly distribute the sample and medium.

• Allow the culture plates to solidify before incubation.

• Incubate the plates at 35–37°C for up to 48 hours for optimal bacterial growth.

• After incubation, visible bacterial colonies will appear.

spreading method

a very simple method to perform bacterial isolation

procedure for spreading method

• The nutrient medium is poured into sterile Petri plates before adding the bacterial sample.

• Allow the nutrient medium in the Petri plates to solidify.

• After solidification, add 1 mL of bacterial suspension onto the surface of the medium.

• Use a T- or L-shaped spreader to evenly distribute the bacterial suspension.

• Incubate the culture plates at 35–37°C for 24–48 hours.

• After incubation, several bacterial colonies will appear.

• The spread plate method is not commonly used for isolating pure cultures.

streaking method

• a very popular and most widely used method for the isolation of pure culture

• limited population of bacteria in the streaking method, pure culture isolation is quite easier than the pour plate and spread plate method

• we can culture, isolate, and study the individual colony of bacteria

procedure for streaking method

• Pour freshly prepared nutrient agar into sterile Petri plates and allow it to solidify.

• Sterilize the inoculating loop by heating it over a flame until red hot.

• Using the sterilized loop, collect the inoculum and streak it over the solidified nutrient agar while keeping the plate near the flame to prevent contamination.

• Incubate the streaked culture plates at 35–37°C for 24–48 hours.

serial dilution method

• known for the isolation and culturing of bacteria

procedure for the serial dilution method

• Prepare serial dilutions of the bacterial suspension in successive test tubes.

• Transfer 1 mL of the sample sequentially to each test tube in the dilution series (10⁻¹, 10⁻², 10⁻³, etc.).

• Inoculate the diluted samples using one of the three methods: pour plate, streak plate, or spread plate.

• Serial dilution makes it easier to isolate bacteria from a smaller bacterial population.

• More concentrated samples (10⁻¹) produce more colonies, while more diluted samples (10⁻⁴) produce fewer colonies.

• Less diluted samples have a higher bacterial concentration, whereas more diluted samples have more water than bacteria.

• Samples with fewer bacteria yield fewer colonies, and chosen colonies are stained and examined under a microscope for identification.

microscope

• most important tool in microbiology research and studies

bright field microscopy

dark field microscopy

phase contrast microscopy

fluorescence microscopy

electron microscopy

parts of a microscope

1. arm

2. base

3. ocular / eyepiece

4. body tube

5. revolving nosepiece

6. objective lenses

7. stage

8. stage clips

9. diaphragm

10. light source / illuminator

11. coarse adjustment knob

12. fine adjustment knob

arm

supports the body tube and connects it to the base

base

serves as the microscope’s foundation and provides stability

ocular / eyepiece

the lens looked through to see the specimen

body tube

holds and aligns the eyepiece to the objective lenses

revolving nosepiece

holds the objective lenses and allows you to switch between them

objective lenses

provide different levels of magnification for viewing the specimen

stage

the platform where the slide is placed

stage clips

holds the slides in place

diaphragm

controls the amount of light that reaches the specimen

light source / illuminator

provides light to make the specimen visible

coarse adjustment knob

moves the stage up and down for focusing

fine adjustment knob

adjusts the focus precisely for a clear image

scanner objective lens

• red

• 4x magnification

low power objective (LPO) 

• yellow

• 10x magnification

high power objective (HPO) 

• blue

• 40x magnification

oil immersion objective (OIO)

• white

• 100x 

• uses oil:

  • cedar oil

  • mineral oil

• has the same refractive index as the mirror

colony form

• overall shape of the colony

• circular, irregular, filamentous

colony edge/margin

• the structure of the colony on its edges that is exposed to air

• entire, undulate, filiform, lobate, curled, scalloped, and serrated/erose

• one type of colony that has a distinct filiform edge is Bacillus anthracis

colony elevation

• defined as the shape displayed by a colony when overserved from the side

• flat, raised, convex, pulvinate, umbonate, crateriform

colony size

• important in fungal and bacterial colony morphology

• described in millimeters

• punctiform, small, medium, large

colony chromogenesis / color / pigmentation

• a colony can display a singular color but have different tones and hues from the middle to the outer part

• the color seen on an agar plate may not be the actual color of the bacteria but the pigment that is produced due to the immersion of the bacteria in the media

• it is crucial to note the opacity of an observed colony 

  • transparent, translucent, opaque, or iridescent

colony surface and consistency

• texture and consistency of the bacteria

• surfaces are described as shiny, dull, smooth, rough, veined, glistening, wrinkled, etc. 

• consistency or texture us often described after touching or scraping on the colony

  • dry, brittle, mucoid, butyrous, viscid

hemolysis

on blood agar, the pattern of red blood cell lysis around the colony

• can be complete = beta-hemolysis

• can be partial = alpha-hemolysis

growth media / media

• substances where microbes are grown

• provides the nutrients necessary to sustain the metabolic activities and reproduction of the microbes

• can be liquid or solid

broth

• liquid media

• used to determine growth patterns in a liquid medium

• the method of choice for growing large quantities of bacteria

agar

• solid growth media

• a mixture of polysaccharides derived from red algae

• solidification agent 

  • it id not broken down by bacteria

  • contains no nutrients that can be used by bacteria

  • melts at high temperatures

• agar plates, agar slants, agar deeps

stocks

• because of the relatively small tube opening and the surface area for growth, agar slants are commonly used and store in bacteria for intermediate period of time

pure culture

a culture that contains a single microbial species

• when left unintended, the culture becomes contaminated

aseptic technique

• a collection of procedures and techniques designed to prevent the introduction of unwanted organisms into a pure culture or into the lab environment

meaning of aseptic

without contamination

sterilization

complete removal of all vegetative cells, endospores, and viruses

• all media which the cells are grown in are sterilized by an autoclave

autoclave

• uses moist heat/steam under pressure to destroy all life forms

• most vegetative cells can be killed at temperatures between 60 to 80 C while bacterial spores require temperatures above boiling 

• needs at least 20 minutes to kill all spores as well as vegetative cells

disinfection

• the killing or growth inhibition of vegetative microbes

• chemical disinfectants (chlorine, bleach, etc.) are used to clean non-living surfaces 

antiseptics

• antimicrobial chemicals safe for use on living skin or tissues 

• example: hydrogen peroxide and isopropyl alcohol 

solid media

• for isolation of bacteria as a pure culture on a solid medium 

• Robert Koch

• agar is used for hardening the media at 1.5-2.0% concentration

• allows the growth of bacteria as colonies by streaking on the medium 

• solidified at 37 degrees Celsius

• growth of bacteria on solid mediums appear smooth, rough, mucoid, round, irregular, filamentous, punctiform

• examples: 

  • nutrient agar

  • MacConkey agar

  • blood agar

  • chocolate agar

semi-solid media

• shows the motility of the bacteria and the cultivation of microaerophilic bacteria

• has an agar concentration of 0.5% or less

• has a jelly consistency 

• the bacterial growth in semi-solid medias appear as a thick line in the medium 

  • examples: 

    • Stuart’s and amies media

    • hugh and leifson’s oxidation fermentation medium

    • mannitol motility media

liquid media

• shows the growth of a large number of bacteria

• broth that allows bacteria to grow uniformly with turbidity

• growth occurs at 37 degrees Celsius in an incubator for 24 hours

• for fermentation studies

• bacterial growth in liquid medias — turbidity is seen at the end of the broth

• examples: 

  • nutrient broth

  • tryptic soy broth

  • MR-VP broth

  • phenol red carbohydrate broth

basal media

• enhances thee growth of many microorganisms

• routinely used medium in the lab, having carbon and nitrogen

• allows the growth of non-fastidious bacteria without any enrichment source

• Staphylococcus and Enterobacteriaceae grow in this media 

• Nutrient agar and peptone water

enriched media

• requires the addition of other substances life blood, egg, or serum

• allows the growth of devised microorganisms but inhibits other and fastidious microbes grow as they require nutrients like vitamins and growth-promoting substances

• blood agar, chocolate agar, LSS, monsor’s taurocholate, lowenstein jensen media

selective media

• shows the growth of selective microbes or desired microorganisms 

• inhibits the growth of unwanted microbes

• inhibition occurs by adding bile salts, antibiotics, dyes, and pH adjustments

enrichment media

• a liquid media that enhances the growth of desired bacteria even at a low density while inhibiting unwanted bacteria

• isolation of soil or fecal microorganisms

• F-broth (salmonella typhi from fecal samples) 

indicator or differential media

• contains indicators that show visible changes to differentiate bacteria based on biochemical reactions

• examples:

  • mannitol salt agar = yellow colonies —> mannitol fermenters

  • blood agar = distinguishes hemolytic vs. non-hemolytic bacteria

  • macConkey agar = pink colonies —> lactose fermenters (pale = non-lactose) 

transport media

• maintains the viability of microorganisms during transport without allowing their multiplication

• examples: 

  • stuart’s trasnport medium - lacks nutrients to prevent overgrowth

  • cary-blair medium - for fecal samples (cholera) 

  • pike’s medium - for streptococci from throat samples

storage media

• used to maintain and preserve bacterial cultures for long periods

• examples

  • cooked meat broth

  • nutrient agar egg saline

aerobic media

supports the growth of microorganisms that require oxygen

anaerobic media

supports the growth of anaerobic bacteria by maintaining low oxygen levels

assay media

used to test potency of vitamins, amino acids, or antibiotics

minimal media

a defined medium with minimal nutrients required for growth of wild-type microorganisms

• used to differentiate wild-type from mutant cells and selects recombinants

fermentation media

• provides nutrients for microbial growth and production of fermentation productions

• components:

  • major: carbon and nitrogen

  • minor: minerals, vitamins, buffers, anti-foaming agents, etc.

resuscitation culture media

specialized medium for reviving stressed or injured bacteria that have lost the ability to grow under normal conditions

preparation of a bacterial smear

• Label the slide – Use a grease pencil to mark one end of the slide with the bacterial culture name.

• Prepare the sample – Place a drop of normal saline on a clean slide and add a small amount of bacteria (from solid or broth culture).

• Spread the smear – Evenly spread the bacteria to form a thin layer, leaving space on all sides for viewing.

• Air dry – Allow the smear to dry at room temperature (25–28°C) in a dust-free area.

• Heat-fix the smear – Pass the slide (smear side up) through a flame three times or use 70% alcohol for 2 minutes to fix the bacteria.

• Avoid overheating – Overheating may burn or distort cells, leading to poor staining results.

• Stain the smear – Apply an appropriate stain on the fixed smear using a staining rack, depending on the staining technique used

Gram staining

• Hans Christian Gram in 1884

• differentiate bacteria into gram positive and gram negative groups based on their cell wall structures 

• some bacteria retain the primary stain (usually crystal violet) due to their thick peptidoglycan layer

• other bacteria lose the primary stain during decolorization and show the counterstain (safranin = red) 

gram staining procedure

• Crystal violet – Primary stain; colors all cells purple.

• Iodine – Acts as a mordant to fix the dye.

• Alcohol (ethanol) – Decolorizer; removes stain only from Gram-negative cells.

• Safranin – Counterstain; colors Gram-negative cells pink.

results from gram staining

• Gram-positive bacteria

  • Thick peptidoglycan layer, no outer membrane.

  • Retain crystal violet and appear purple.

• Gram-negative bacteria

  • Thin peptidoglycan layer, has an outer membrane that dissolves in alcohol.

  • Lose crystal violet, take up safranin, and appear pink/red.

acid-fast staining (ziehl-neelsen method)

• used to identify acid-fast bacteria that cannot be stained by the gram stain due to their waxy cell wall

• Mycolic acid makes the bacterial cell wall waxy and impermeable to most stains.

• Heat softens the wax, allowing the primary dye (carbol fuchsin) to enter the cells.

• Acid-alcohol is used as a decolorizer:

  • Acid-fast cells resist decolorization and retain the red dye.

  • Non–acid-fast cells lose the dye and are later stained blue with methylene blue

acid-fast staining procedure

• Primary stain: Carbol fuchsin (red)

• Heat: Helps the dye penetrate the waxy cell wall

• Decolorizer: Acid–alcohol

• Counterstain: Methylene blue

results from acid-fast staining

• Acid-fast bacteria (AFB): Bright pink/red (retain carbol fuchsin)

• Non–acid-fast bacteria: Blue (take up methylene blue)

endospore stain

• endospores = dormant, non-reproductive structures formed by gram-positive bacteria

• allows the bacteria to withstand harsh environmental conditions

• makes endospores visible under a bright background since they resist normal stains

endosporulation

• DNA replication occurs in the bacterial cell.

• Layers of peptidoglycan and protein form around the DNA.

• The endospore matures and is released from the cell.

• It can remain dormant for years until conditions become favorable again.

• When conditions improve, it germinates into a vegetative (active) cell.

endospore staining procedure

• Primary stain: Malachite green (water-soluble; penetrates spores with heat).

• Heat: Acts as a mordant to drive the dye into the spore coat.

• Decolorizer: Water removes the dye from vegetative cells.

• Counterstain: Safranin stains the vegetative cells pink/red

result for endospore staining

• Endospores: Green

• Vegetative cells: Pink/red

how to solve vsp/cmu

what if the given plated medium is only 0.1 ml?

• multiply by 10

• multiply to the reciprocal of the test tube it came from

what if the given plated medium is only 0.2 ml?

• multiply by 5

• multiply to the reciprocal of the test tube it came from

culture

increase in the population of bacterial cells

binary fission

• the prevailing means of bacterial reproduction

• division exactly in half

• most common means of bacterial reproduction

  • forming two equal size progeny

  • genetically identical offspring

  • cells divide in a geometric progression doubling cell number

• doubling time is the unit of measurement of microbial growth

generation time

the time it takes cells to divide and double in population

generation time of Escherichia coli

15-20 minutes

generation time of Staphylococcus aureus

20-30 minutes

generation time of Salmonella enteritidis

20-30 minutes

generation time of Mycobacterium tuberculosis

15-20 hours

generation time of Lactobacillus acidophilus

66-67 minutes