Microbiology Lab Exam 1

Occupational Health Care provider

call if you have health concerns or questions

Questions about chemicals can be answered by referring to the

appropriate Material Safety Data Sheet (MSDS)

first unexcused absense deduction

5 lab points

second unexcuse absense deduction

10 lab points

3rd unexcused absense deduction

zero for your lab

receive an unexcused absense for reasons including, but not limited to:

• 3 Tardies (arriving within 10 minutes of the start of lab according to the lab clock)

• arriving more than 10 minutes after the lab period has begun (you will not be allowed to enter the lab at this time)

• Leaving before all experiments are complete

must schedule a make-up at least how many hours before leaving on a University excused field trip or interview

72 hours

• all other excused absences should be schedules within 48 hours of the absence and completed within 5 working days

Total absences that exceed 20% of scheduled lab meetings may result in an

F in the course

For excused absences you must get an excuse from

Lacy Basile

• complete the make-up request form, link on canvas, include course and section number

Glass microscope slides 🡪

Glass disposal

Glass tubes with media (sterile or not/broken or not) 🡪

Biohazard bin

Pipette tips 🡪

Biohazard waste

Paper towels 🡪

Regular trash

Microorganisms are

“ubiquitous in nature”

• Found everywhere that other lifeforms exist

  • Have a long, rich history on Earth

  • And have successfully adapted to a wide range of habitats

Microorganisms can be isolated from

• Soil (all kinds!)

• Water (over a large range of salinity)

• Plants

• Animals (even humans!)

• Even seemingly uninhabitable places such as the hot acid pools in Yellowstone (approx. 85ᵒC with a pH near 1)

• Does NOT apply to EVERY SPECIES, but certainly applies to microorganisms as a group

Classification of microorganisms

• Many microorganisms do not reside on or in a specific plant or animal host and are not known to cause disease

  • Free-living and nonpathogenic

  • Often saprophytes that perform important role of decomposition in the ecosystem

Other microorganisms reside on or in a host organism

• pathogens

• mututalism

• commensals

pathogens

cause damage to host

Mutualism

both host and microbe benefit

Commensals

microbe benefits but no effect on host

Many of the commensal /mutualistic strains are

opportunistic pathogens

• Inhabit our bodies and are capable of producing a disease state if introduced into a suitable part of the body

• reservoir

reservoir

any area where a microbe with the potential to cause infection resides

Purpose of blood agar

To differentiate bacteria based on their hemolytic characteristics

Blood agar

Includes 5% sheep blood in a Tryptic Soy Agar base

Several species of Gram-positive cocci produce exotoxins called

hemolysins, (streptococci produce streptolysins) which are able to destroy RBCs and hemoglobin

Three major types of hemolysis:

• β – hemolysis

• α – hemolysis

• γ – hemolysis

β – hemolysis

complete destruction; results in clearing of the medium

• yellow glow

α – hemolysis

partial destruction; results in greenish discoloration of the medium

• brownish/green

γ – hemolysis

no hemolysis

• clear/blue

Germicides

• Refers to substances or systems, both chemical and physical, that prevent the spread of pathogens

• Some are specific but most target a broad-spectrum

Three categories of Germicides

• Decontamination

• Disinfection

• Sterilization

Decontamination

• Lowest level of control

• Defined as “reduction of pathogenic microorganisms to a level at which items are safe to handle without protective attire”

• Usually includes physical cleaning with soaps or detergents, and removal of all or most organic and inorganic matter

Disinfection

• Next level of control

• Divided into three sublevels – low, medium and high – based on effectiveness against specific control pathogens or their surrogates

  • All kill most, if not all, of the targeted pathogens but typically do not kill large numbers of spore

  • Typically liquid chemical agents but can be solid of gaseous

  • Other methods include dry heat, moist heat, and ultraviolet light

Types of disinfectants

• Chemical sterilants

• Antiseptics

Chemical sterilants

High-level disinfectants that have the ability to kill all vegetative cell and some spores

Antiseptics

Disinfectants designed to reduce or eliminate pathogens on or in living tissue

Sterilization

• Highest level of pathogen control

• Complete elimination of viable organisms including spores

• Can be achieved by some chemicals and gases, incineration, dry heat, moist heat, ethylene oxide gas, ionizing radiation, low-temperature plasma, or low-temperature ozone

Steam Sterilization

• Most effective and most common method

• Autoclave

Autoclave

• Device most commonly used for steam sterilization

• Use superheated steam under pressure to kill heat-resistant organisms

In the microbiology laboratory:

• Sterilizing temperature is set between 121°C and 127°C

• Must reach optimum temperature for a least 15 minutes

• Time varies according to the size and consistency of the material

• Color-coded autoclave tape

• Biological indicators

Biological Indicator

• Indicator vial includes:

  • Small ampule containing fermentation broth with pH indicator

  • Strip of filter paper containing bacterial spores

Test:

• Vial is autoclaved at 121°C for 15 minutes

• Ampule is crushed

  • Allows fermentation broth to come into contact with bacterial spores

• Vials are incubated at 55°C for 48 hours

Biological indicators are the only way,  with certainty, to determine that

sterilization has been achieved

Acidic condition from fermentation

right colored indicator

Negative Control(not crushed)

Ubiquity of Microorganisms - Procedure

• Day 1

  • You will transfer microorganisms from seemingly uninhabited sources and grow them on agar plates

  • When you inoculate the plates, you will transfer an unknown number of unknown cells 

  • All cells that are able to grow on the plate will divide and produce visible colonies

  • Each source will likely have multiple species present representing a mixed culture

• Day 2

  • You will examine various growth characteristics produced by these “invisible” cohabitants

  • If a colony is not contacting any other colonies, it is said to be isolated and can be transferred to a sterile medium to start a pure culture 

    • Consider mixed vs. pure culture

 isolated

If a colony is not contacting any other colonies

• can be transferred to a sterile medium to start a pure culture

writing vs typing organism names

typing will be italisized and writing will be underlined

What does aseptic transfer mean?

Transfer of living microbes from one place to another without contamination of:

• The culture

• The sterile medium

• Or the surroundings

To prevent contamination

Inoculating instruments must be sterilized prior to use

• Wire loops are sterilized immediately before use in an incinerator

• Cool loop in agar before transfer

• Mouths of tubes containing culture or media are also incinerated at the time of transfer

• Instruments that can not be incinerated are sterilized inside wrappers or containers by autoclaving prior to use

What is a culture?

A medium that contains living microbes

• A pure culture is one that contains a single species

• A mixed culture is one that contains multiple species

  • All cultures are assumed to be mixed prior to obtaining isolation!

Microbial cultures are grown and maintained on or in solid and liquid substances called

media

Media come in many forms, each with a specific application

• Broths are used to grow microbes when fresh cultures or large number of cells are required

• Agar slants are generally used to grow stock cultures that can be refrigerated after incubation and maintained for several weeks

• Plated media are typically used for obtaining isolation of species, differential testing, and quantifying bacterial densities

A bacterial sample is ALWAYS assumed to be a

mixed culture

Mixed culture

microbial culture consisting of two or more species

Pure culture

microbial culture containing only a single species

Methods of isolation

• Streak Plate

• Pour plate (Lecture Only)

• Spread plate

In the 1850’s, Louis Pasteur developed concept of the

pure culture

• Allowed microbiologists to study individual species

• His methods of isolation were terribly inefficient

  • Tried to dilute cultures until he thought he had a single cel

In the 1880’s Robert Koch developed a procedure for

“streaking for isolation”

The primary purpose of streaking for isolation is to

obtain isolated colonies

• Can then be used to obtain a pure culture

  • A single colony (composed of identical cells) is then used to inoculate another culture 

An isolated colony is composed of

millions of cells – all identical and all descendants from a single cell or group of cells

In the quadrant streak method:

• A bacterial sample is streaked over the surface of a plated agar medium.

• During streaking, the cell density decreases, eventually leading to individual cells being deposited separately on the agar surface

• This results into a colony consisting only of the original cell type

• COMMONLY USED TECHNIQUE: QUADRANT STREAK 

Quadrant Streak

Used with samples suspected of high cell density

Quadrant Streak Method 2

Quadrant Streak Method 3

Properly streaked plate with isolated colonies in the 4^th quadrant

Result of not sterilizing loop between quadrants

Improperly streaked quadrants

Result of not rotating plate properly

Either loop was not cooled or loop did not pass through previous quadrant

Colony Morphology

One cell makes two, two make four, four make eight…one million make two million

• Eventually, a visible mass of cells known as a colony appears

• Color, size, shape, and  texture are determined by the genetic makeup of the organism

Growth characteristics of colony morphology are influenced by:

• Nutrient availability

• Temperature

• Incubation time

colony morphology shapes

• Round

• Irregular

• Punctiform (tiny pinpoint dots)

colony morphology Margin

• Entire (smooth, with no irregularities)

• Undulate (wavy)

• Lobate (lobed)

• Filamentous (unbranched strands)

• Rhizoid (branched like roots)

colony morphology Elevation

• Flat

• Raised

• Convex

• Pulvinate

• Umbonate

colony morphology Texture

• Moist

• Mucoid

• Butyrous

• Dry

• Shiny

• Dull

• Other properties include color (pigment production) and optical properties (opaque and translucent)

Agar slants are primarily used for

cultivation, maintenance, and storage of stock cultures

Organisms cultured on slants display a variety of growth characteristics:

• Filiform

• Friable

• Spreading edge

• Pigmented or translucent/transparent

Filiform

dense and opaque with a smooth edge

Friable

crusty

Spreading edge

produced by motile organisms

Microorganisms culture in a broth also display

characteristic growth patterns

• Pellicle

• Sediment

• Uniform fine turbidity

• Flocculent

Pellicle

Growth floats on top of the medium

Sediment

Growth sinks to the bottom

Flocculent

Clumped growth

What is the Pour Plate Technique

Yields isolated colonies of bacteria and fungi

• Original sample volume is diluted several times to reduce microbial population and to calculate original sample concentration

• Small volumes from each dilution are mixed with molten Tryptic Soy Agar (TSA) (48-50C) in sterile petri dishes

• After the agar has solidified, each cell will be fixed in place and form a colony

• The total number of colonies can then be used to calculate the number of viable microorganisms in the sample

  • Calculated as CFU/mL (colony forming units/mL of sample)

What is the spread plate technique?

• A method of isolation in which a diluted microbial sample is deposited on an agar plate and spread uniformly across the surface

• If properly diluted, cells (colony forming units/CFUs) will be deposited far enough apart on the agar surface to grow into individual colonies

After incubation, a portion of an isolated colony can be transferred to a sterile medium to begin a

pure culture

• Also can be used to quantify cell density of a broth culture

Perform a serial dilution of a broth culture

• Take 10 μL of undiluted culture, add that to a tube containing 990 μL of sterile H₂0, and gently mix/vortex. (Tube A)

• Then take 100 μL from tube A and transfer to another tube containing 900 μL of sterile H₂0, and gently mix/vortex. (Tube B)

• Continue this process of transferring samples from higher dilutions to sterile water to create more and more diluted samples, hoping to get a plate that will have the right number of colonies to be considered “countable” (30-300)

• Transfer and spread 100 μL of each dilution on a TSA plate. 

• Lastly, incubate and count colonies

How is the spread plate technique useful?

• Achieve isolation

  • May be used to start a pure culture

• Estimate cell density

  • CFU/mL =              # colonies          .     

                    volume plated x dilution

Calculating Dilutions for Spread Plate

• 1^st tube - Dilution = Volume transferred/total volume of tube 

  • 10μL/(10μL+990μL) = 10/1000 = 0.01 = 10^-2 (Tube A)

• 2^nd tube – Dilution =  concentration from the previous tube x (volume transferred/total volume)

  • 10^-2 x (100μL/100μL+900μL) =  0.001 = 10^-3

• 3^rd tube – Same as the second tube calculation

  • 10^-3 x (100μL/100μL+900μL) = 0.0001 = 10^-4

Bright-field microscopy

Produces an image made from light transmitted through a specimen

• Specimen restricts light transmission and appears dark against a light background

• Because most biological specimens are transparent, contrast is improved with the application of stains to the specimen

  • Staining process usually kills cells

Image formation of microscope

• Begins with light from an internal or external light source

• Light passes through the condenser, which concentrates the light, then through the specimen and the objective lens

• As the light passes through the objective lens it is refracted, or bent, and produces a magnified “real image”

• The image is magnified again as it passes through the ocular lens to produce a “virtual image”

Total Magnification   =  

Magnification of ocular lens  X    Magnification of objective lens

Immersion oil will ONLY be used on the

100x objective

• Other types of microscopy

• Dark-field microscopy

• Phase contrast microscopy

• Fluorescence microscopy

Dark Field

Light travels in a path past the objective unless scattered into it

Phase Contrast

•Scattering produces 2 changes in light: change in brightness and change in phase.

•Human eye is only sensitive to changes in brightness

•Phase contrast microscopes make phase shifts visible as differences in brightness

Fluorescence

•High energy light (usually a laser) outside the visible spectrum strikes the sample

•Any fluorescent molecules absorb that light and emit lower energy, visible light back to the viewer

How are bacterial stains useful?

They allow us to see cellular morphology which include:

• Size (length, width, diameter)

• Shape

• Arrangement 

• Other structures or differentiating features 

Determining cellular morphology is an important step in identifying a bacterial species