o Staphylococcus aureus could be present/colonise the human

body.

o Incidence of S. aureus in humans:

- persistent carriers; carry one type of strain of the population

(20%)

- intermittent carriers; form the large part of the population (60%)

- non carriers; never carry S. aureus

What is the role of the MEA plate?

Detection, isolation and enumeration of fungi.

Name the organism that is identified using the Bactistaph.

Staphylococcus genus

-----------------------------

o A single colony consists of identical cells and can be used to

provide subsequent pure cultures of that species in broth/agar

media.

o The inoculated area forms the primary inoculum.

Explain why agar is used in preference to gelatin.

Agar is from algae.

Gelatin is from animal bones.

Agar is easier to extract and it has a higher melting point.

Why do well-separated colonies appear larger than those in areas of

heavy growth?

More nutrients available.

Explain the purpose of passing the dried smear through the Bunsen

flame

Kill the cell, and make it stick to the slide. Alters the cells so it can

readily accept the stains/dyes.

Suggest the effect on the gram stain if a whole colony is used to make

the smear.

If you use too many cells, you won’t be able to see as well, or even at

all. You want a few cells, so that you can see the cells clearly, not

because the result will be different.

Which structure of the bacterial cell determines its gram reaction?

Peptidoglycan cell wall determines whether the cell wall gets stained

or not.

Explain which technical step of the Gram-staining procedure is crucial

in determining the outcome.

Alcohol.

Over; + = purple, - = pink

Under; - = pink, + = purple

gram + ; retain primary stain (thicker)

gram - ; destain (thinner)

gram + ; violet stain is trapped by peptidoglycan layer which forms the

outer layer of the cell.

gram - ; Outer membrane prevents the stain from reaching the

peptidoglycan layer. The outer membrane is permeabilised by alcohol

and the safarin stain is trapped by the peptidoglycan layer.

-----------------------------

o Each bacterial species possesses characteristics by which it can

be fully identified even to subspecies.

o The visible characteristics are termed morphology.

E. Coli Klebsiella sp. P. vulgaris

Gram – rods. They don’t form spores and can live in the

presence/absence of oxygen.

Why do some organisms form clusters and others chains?

Bacteria reproduce rapidly. It is in their nature to split apart when they

reproduce. In reproducing themselves, they have no time to roam

around. Bacteria that multiply quickly and have motility form colonies

into clusters.

Describe the shape and arrangement of endospores. Why are they

difficult to stain?

Stains can’t penetrate the spore covering (exosporium). You have to

heat it to open it than stain it. Also, due to its low permeability and high

degree of resistance due to multiple coats surrounding the spore. We

want to stain them to see if the bacterium cells have highly resistant

spores within their vegetative cells.

What advantage might motility confer on a microorganism?

Bacteria can move toward greater concentration of food, and away

from greater concentration of waste products.

What advantage does the capsule confer on a potential pathogen?

Capsules make the bacteria more resistant to harsh conditions, and self

protect themselves from antibiotics.

-----------------------------

o The normal microbial flora of the human body consists of

organisms, which have adapted to the physic-chemical

conditions of their particular habitat.

o Resident flora – where organisms consistently are found at a

given site and multiply.

o Resident flora reduces the possibility of invasion by potential

pathogens by occupying attachment sites and successfully

competing for nutrients.

o Opportunistic pathogens – gain access to another site.

o Transient flora – species temporarily found at a given site but

do not multiply there.

o Transient skin flora is of particular significance because they

are frequently implicated in transmission of infection.

o Nosocomial infection – outbreaks of S. aureus in a hospital

environment due to poor hand washing practices.

-----------------------------

o Bacterial endospores are recognized as being thermoduric; they

can survive prolonged exposure to 100 degrees.

o Moist heat, using steam at elevated temperature and pressure in

a sealed chamber. The process depends on the steam being

saturated, total removal of air, all surfaces being accessible to

steam and the conditions being maintained for sufficient time.

(121 degrees for 15 minutes at 101 kPa).

What properties of steam make it an ‘ideal’ sterilent?

Rapid, efficient and non-toxic.

It kills cells by denaturing their components resulting in the loss of

function and death.

Moist heat is very efficient as it destroys the microorganisms by

disintegrating their nucleic acids and cell membrane, as well as

denaturing both the enzymes, and the important proteins required for

the microbe to function competently

Compare the biocidal action of moist and dry heat.

The biocidal action of moist heat causes proteins to denature and

coagulation occurs. Whereas, the biocidal action of dry heat dries the

cell up, which causes membrane fusion.

List the various stages that make up the total sterilization cycle.

The sterilization cycle comprises of two stages - the penetration and

holding time. The penetration time ensures that the temperature

required to destroy microorganisms of 121°C is met, while, the holding

time maintains the temperature of 121°C for 15 minutes. If the process

was operated appropriately, it is able to destroy all microorganisms; as

well as bacterial spores that are known to be highly heat resistant.

Explain the term tyndallization.

the process of destroying vegetative bacteria with the use of steam.

Unlike steam sterilization, tyndallization involves the bacteria being

exposed to steam for a total of three times repeatedly and incubating

them for 24 hours between every exposure to steam.

Apart from heat, discuss other physical control methods available.

Some physical control methods available are filtration and ultra violet

radiation. Filtration is a method used to reduce the population of

microbes in solutions of materials that are sensitive to heat. It is used to

sterilize different liquids and gases, as well as air. UV Radiation on the

other hand, is used to inhibit the occurrence of replication and

transcription. This method is not as efficient due to its disadvantage of

being a sterilizing agent in limited situations.

Can prions be destroyed by normal steam sterilization process?

Prions are very difficult to destroy as they are resistant to heat, radiation,

as well as many other factors that are capable of destroying a

microorganism. As they are mainly composed of proteins, the normal

sterilization process of 121°C for 15 minutes is not effective against them,

due to the high temperatures required to denature the proteins.

Sterilizing will only work against prions is if the protein is denatured to the

point where they are unable to produce the abnormal folding of

normal proteins.

-----------------------------

o Microorganisms have optimum requirements for active growth,

but can otherwise tolerate a wide range of conditions.

o The only limits to survival are extremes of temperature, lack of

moisture and the presence of substances that are toxic to the

cell.

o 4 environmental influences – temperature, hydrogen ion

concentration, water activity and oxygen availability.

-----------------------------

o Differential medium – An indicator system can be incorporated

into the medium to differentiate the desired organism from

others which may also grow.

o Selective media – those using an inhibitory agent to suppress the

growth of unwanted species.

o MacConkey agar is both selective and differential. It is

differential in that the lactose fermenting bacteria appear as red,

pink or purple, and the non-lactose fermenting bacteria

produces colourless and translucent colonies or beige or even

green/brown.

o The media could be made selective by the addition of different

types of bile salts.

o MacConkey agar consists of:

- Nutrient base (2% peptone); peptone = beige/clear colonies

- Bile salts; inhibits all non enteric bacteria but selective for

various enteric bacteria

- Lactose; break down to lactic acid = red/dark purple colonies

- Neutral red indication – pH indicator; detects acid end

products produced by fermentation of lactose lowering the pH,

the indicator turns red. Detects ammonia produced by the

deamination of amino acids in peptone raising the pH, the

indicator turns cream/beige colour.

o Enriched media enhance basal media to improve the growth of

the desired organism. This can encourage prolific growth of

unwanted species so selective agents are often included

making an enriched, selective medium.

Explain why you would inoculate the nutrient agar before the

MacConkey agar (w/o salt-NaCl) and then 3 if the same loopful of

culture is used.

MacConkey agar is both selective and differential. It contains bile salts,

and the dye crystal violet, which inhibit the growth of gram + bacteria

and select for gram – bacteria.

Explain why the MacConkey plates should not be incubated for more

than 24 hours.

Bacteria starts utilizing the peptone so we won’t be able to

differentiate.

Discuss the selective and differential basis of the MacConkey agars

used, including the appearance of lac+ and lac- colonies.

lac+ = pink colonies, lac- = beige/yellow.

Basis differentiation – lactose fermentation.

-----------------------------

o Quantitative determination of microbial populations is essential

to many lab procedures.

o 3 techniques are widely used – viable count, total cell count and

turbidometry.

o Viable count – provides information about the population

density of living cells only.

o Total cell count – counting chamber to obtain an accurate

count of the total number of cells of all types in a known volume

of liquid.

o Turbidometry – uses the level of turbidity measured by

spectrophotometry to estimate the biomass.

o Colony forming unit – possibility that a single colony may have

arisen from two or more bacterial cells in close proximity.

Discuss why duplicate plates of 3 consecutive dilutions are used to

obtain colony counts. Would you always use the same dilutions?

Reduce human errors.

Explain why the viable count is expressed as cfu ml rather than cells ml.

1 cell = 1 colony

Why is spread plate method preferable to the pour plate?

Spread plate – easier to count. Volume = o.1 ml

Pour plate – small colonies and embedded in agar.

Why is a washed suspension used to determine the dry mass instead of

an untreated culture?

There’s a fixed amount.

Discuss the major sources of error in this method for determining cell

mass.

- dead cells

- extracellular material

-----------------------------

o Bacteria are typically grown in batch culture – a close system

with a finite supply of nutrients and oxygen, which will gradually

be exhausted.

Predict what would happen if the starting culture which was added to

the growth medium was:

An old culture: An old culture contains old cells where they have

depleted necessary nutrients that are required for division to occur. The

addition of an old culture to a growth medium causes the lag phase to

occur in a growth curve. Time is required for the old cells to recover, as

they may have been damaged previously. Time is also needed for ATP,

essential cofactor and ribosomes synthesis. Furthermore, the previous

medium may have a different composition to the BHI medium, and so,

the cells must produce new enzymes that are suitable for the BHI

medium.

In exponential phase: If a culture in exponential phase was added into

a growth medium, an unbalanced growth occurs. This is due to the

difference between the nutrient levels and environmental conditions

from the previous medium to the new growth medium. New suitable

nutrients and cellular constituents must be synthesized until equilibrium

is reached. There are two types of experiments where unbalanced

growth can be observed: shift-up or shift-down. Shift up is essentially

where a culture is transferred from a nutritionally deprived medium, into

a richer medium. While shift-down is where a culture is transferred from

a nutritionally rich medium, into a deprived medium. When a culture is

in a shift-up experiment, it must enter lag phase where they synthesise

new ribosomes and nutrients required for the cells to grow and divide,

before entering the exponential phase where balanced growth is

recommenced.

Suggest reasons to explain the stationary phase.

There are several factors to why microorganisms enter the stationary

phase. When the necessary nutrients become depleted, population

growth decreases. Oxygen availability is another factor. Oxygen is

soluble to a certain extent, thus it has a tendency of becoming

depleted rapidly. If this occurs, the cells under the surface are unable

to grow and divide, unless the culture is placed in an aerated

condition. Toxic wastes accumulating may also cause a decrease in

population growth

Discuss whether the doubling rate of the organism used is typical of

most bacteria.

The mean generation time for the aerated condition is 41.67 minutes,

whereas for the non-aerated condition, it is 39.48 minutes. This indicates

that when Vibrio natriegens doubles much faster in an aerated

environment, compared to a non-aerated environment. This is due to

the facultative anaerobe nature of Vibrio natriegens.

Explain why the growth and recovery media are supplemented with

NaCl.

The bacterium Vibrio natriegens is a gram negative, non-pathogenic

marine bacterium that is able to grow extremely. It is a halophilic

bacterium, thus approximately 2% of NaCl is a requirement for it to

grow. With the presence of sodium ions, V. natriegens reacts well, as

the sodium ions promote their growth. NaCl acts as a supplement for

the growth of Vibrio natriegens and recovery media as a cell is unable

to survive, and thus grow where water is in a pure form.

Discuss the effect of aeration on Vibrio Natrigens.

The bacterium, Vibrio natriegens is a facultative anaerobe, halophilic

marine bacterium. Facultative anaerobes are able to survive in the

presence or absence of oxygen. Due to aerobic respiration, they are

able to produce ATP in oxygen present conditions. However, in the

absence of oxygen, they are able to change produce ATP by the

process of fermentation. Although Vibrio natriegens can live in the

presence of absence of oxygen: being a facultative anaerobe, it

prefers living in an aerobic condition. Therefore, placing Vibrio

natriegens in an aerated environment enables them to grow more

efficiently. While, placing Vibrio natriegens in a non-aerated

environment, the population growth will still increase, but to a lesser

extent.

-----------------------------

o Genotype – complete genetic composition of the DNA and

determines all its characteristics such as physical appearance

and metabolic functions.

o Phenotype – observable characteristics

Which temperature results in pigmented colonies?

30 degrees

Explain whether the temperature response is a phenotypic or

genotypic change.

Temperature is a phenotypic response.

Suggest a possible mechanism to explain the relationship between

pigmentation and incubation temperature.

Why is it necessary to make serial dilutions of the culture for UV

radiation?

Explain whether irradiation results in a phenotypic or a genotypic

change.

Discuss possible ways by which a single mutation could produce this

effect.

-----------------------------

o Prototrophic bacteria are able to synthesise all their nutritional

requirements from simple starting materials.

o When exposed to a mutagen, one of the possible outcomes is

the emergence of individual cells, which have lost the ability to

synthesis or utilize a specific intermediate in a metabolic

pathway.

o Auxotrophs – nutritionally deficient progeny

o After exposing the parent strain to a mutagen, the surviving cell

population is recovered and grown in the presence of penicillin.

-----------------------------

o Yeasts grow as colonies, which are very similar to bacterial

colonies in appearance and texture, but usually larger.

o Many fungi can exist in either a filamentous form or a yeast form,

depending on environmental factors – dimorphic.

o The filaments are not true hyphae but single cells joined in chains

– pseudohyphae.

o During filamentous growth, these organisms can produce

blastospores, which are outgrowths along septate hyphae and

chlamydospores within or at the tip of the hyphae.

Describe the major differences between yeasts and filamentous fungi.

Yeasts:

- reproduce asexually

- unicellular

- colourless parasite

Filamentous fungi:

- reproduce sexually and asexually

- multicellular

- colourful parasite

What is the difference between vegetative and aerial mycelia?

Vegetative is the part that is actually in physical contact with whatever

the fungi is feeding on. It is the part that anchors and absorbs nutrients.

Aerial is the part that produces asexual spores.

What is the main purpose of the slide culture technique?

When you do slide cultures, you’re growing the fungi directly on the

slide on a thin film of agar. By doing this, you don’t have to remove a

portion of the fungus from a culture plant and transfer it to the slide, so

there’s less chance for the features that are key to identification,

notably the spore-bearing structures to be damaged.

In general, what temperature do environmental fungi prefer to grow?

16 – 27 degrees.

Discuss the features of fungi that are important in their identification.

Look for where on the cap the stem extends from and what features

are present underneath the cap.

Discuss the presence of C. albicans in the tongue region and the

infection it can cause in humans.

C. albicans is commensal and a constituent of the normal gut flora

comprising microorganisms that live in the human mouth and

gastrointestinal tract. C. albicans lives in 80% of the human population

without causing harmful effects, although overgrowth of the fungus

results in candidiasis. Candidiasis is often observed in

immunocompromised individuals such as HIV-infected patients.

Which sites of the body are most subject to fungal infections?

In damp, dark places.

- toes/toenails

- mouth/tongue

- under stomach fat

- under breasts

- armpits

- groin

- vagina

-----------------------------

o Heterotrophic Plate count – enumeration of viable bacteria is

made by the pour plate method using nutrient agar.

o Tube dilation method – two stage process; first identifies the level

of coliforms present, and stage the level of thermotolerant

coliforms.

o Coliform count – ability of coliform bacteria to produce acid in

MacConkey’s liquid medium at 37 degrees.

o Thermotolerant coliform – confirms E. Coli presence.

o Colilert method – used to test drinking water for the presence of

total coliforms generally and E. coli specifically.

Why is Pseudomonas aeruginosa mentioned specifically in these

standards?

It cannot exist in water samples.

What is the advantage of using MacConkey agar No. 3?

It inhibits all gram + bacteria.

Name 2 protozans that are involved in major outbreaks of waterborne

diseases.

Giardia spp. and Cryptosporidium.

Why is Cryptosporidium a problem for swimming pool operators?

Cryptosporidiosis is an infection, which occurs after the accidental

swallowing of contaminated pool water. It is highly contagious and

highly resistant to chlorine disinfection.

Once a pool is contaminated it can remain a source of infection for

pool users for prolonged periods of time, due to cryptosporidium’s

resistance to chlorine, and difficulty of removing infection through

filtration systems. Pool operators can reduce the risk of initial

contamination by using common sense operating practices.

Discuss the significance of Legionella and Mycobacterium species in

cooling towers.

Legionella causes respiratory tract infections.

Mycobacterium causes tuberculosis

Apart from the microbiological tests, give two common chemical tests

carried out which would indicate if the water is good for drinking.

- pH test

- salinity test

-nitrate test

-----------------------------

o Food may be contaminated with microorganisms from various

sources, initially from the raw materials used, then during

preparation and processing and finally post preparation during

transportation and storage.

What is the most common source of bacteria in milk?

Fresh milk is normally sterile. Contamination occurs with the pipeline or

farmers don’t have enough time.

Enterococcus faecalis; it can withstand pasteurization.

Discuss the significance of antibiotics in milk for the dairy industry.

Antibiotics in milk could cause bacteria resistant to the same antibiotics

used to treat people for infections. If people are exposed to these

bacteria, effective treatments would be harder for doctors to find.

Discuss the significance of lipolytic and caseinolytic microorganisms in

milk and the impact to the dairy farmer.

Lipolytic microorganisms break down milk fat and lead to the

production of non-fat milk.

Caseinolytic microorganisms break down casein and lead to the

production of normal milk, and help produce cheese.

Discuss the presence of Listeria monocytogenes in food and the health

impacts to humans.

Listeria monocytogenes has the potential to be present in all raw foods.

It causes one of the most severe forms of foodborne infection.

---------------------------------------

Malt Extract Agar (MEA)

- high content of peptone

- acidic

- yeast will grow on it, but bacteria will not

- isolation of fungal microorganisms

Horse blood agar (HBA)

- blood enriched microbiological culture media

- as it is enriched, it allows the growth of certain fastidious bacteria, and

allows indication of haemolytic activity

Nutrient Agar (NA)

- used for the growth non-fastidious organisms and observation of

pigment production

MacConkey Agar (MAC)

- selective and differential medium containing lactose, bile salts,

neutral red, and crystal violet.

- bile salts and crystal violet inhibits the growth of gram +

- neutral red dye is a pH indication (colourless = > 6.8, red = <6.8)

- acid accumulating from lac fermentation turns the dye red

- lac fermenters turns red on the macconkey agar whereas lac non-

fermenters remain their normal colour/colour of the medium

- formulations without crystal violet allow growth of enterococcus and

some species of staphylococcus, which ferment the lac and appear

pink on the medium

- selective; bile salts = support the growth of gram -

crystal violet = inhibit the growth of gram +

- differential; neutral red indication as visual response

- selective and differential media used to differentiate gram – while

inhibiting the growth of gram +

- the addition of bile salts and crystal violet to the agar inhibits the

growth of most gram +, making the agar selective

- lactose and neutral red are added to differentiate the lactose

fermenters, which form pink colonies, from lactose nonfermenters that

form clear colonies

Tryptic Soy Agar (TSA)

- general purpose media produced via enzymatic digestion of

soybean meal and casein.

- support growth of many semi-fastidious bacteria

Mannitol Salt Agar (MSA)

- selective and differential media

- mannitol indicates organisms that ferment mannitol: mannitol

fermentation produces lactic acid, lowering the pH and turning the

plate yellow

- The salt is to select for halophiles; organisms that cannot withstand a

high salt content will be unable to grow well

-----------------------------

PLATING

Spread plate

- known volume

- less colonies

- grows on the surface

Pour plate

- known volume

- colonies embedded in agar and on the surface

- more isolated colonies

Streak plate

- unknown volume

- different colonies

- more colonies/close together

-----------------------------

STAINS

Ziehl-Neelsen stain

- mycobacterium and nocardia species

- differential stain used to detect acid and alcohol-fast organisms in

fixed smears

- acid fast organisms stain red

- other organisms stain blue

India Ink

- capsules

- negative stain technique

- does not stain the bacteria, but stains the background, so that the

capsule appears as a clear halo surrounding the cell

Maneval’s stain

- capsules

- capsules are negatively stained

- red; organisms

- grey; background

- clear halo around the organisms; capsule

Neisser stain

- polyphosphate granules; characteristic feature of Corynebacterium

- granules appear blue-black and the cell stains pink

Schaeffer and fulton stain

- endospores (in some gram + and – bacteria)

- spores appear green, while the remainder of the cell and non-sporing

cells stain red

Mast/Hanging drop/Craigie tube method

- motility

Gram stain

- used to differentiate organisms based on the cell wall structure as well

as observing shape and arrangement

- gram + and yeasts stain purple

- gram – stain red/pink

Kinyouns method

- acid-fast bacteria; red

- non acid-fast bacteria and background; green

-----------------------------

GROWTH IN LIQUID MEDIA

Turbidity

- present or absent

- present; slight, moderate, dense

- cloudy

Deposit

- present or absent

- present; slight, moderate, abundant

Surface growth

- present or absent

- present; surface pellicle (surface membrane) or growth around the

bottle

o Shape

o Size

o Texture

o General appearance

o Pigmentation in the medium

Micrococcus luteus - NA

- gram +

- cocci

Streptococcus pneumonia - HBA

- gram +

- cocci

- alpha hemolysis

Clostridium sporogenes - HBA

- gram +

- rods

- beta hemolysis

Proteus vulgaris - NA

- gram -

- rods

Mycobacterium phlei - NA

- gram +

- rods

Klebsiella spp. - NA

- gram -

- rods

Bacillus cereus - NA

- gram +

- rods

E. Coli - NA

- gram -

- rods

- enterobacteriaceae species

Staphylococcus aureus - NA

- gram +

- cocci

Bacillus megaterium

- gram +

- rods

Enterococcus faecalis - gram +

- cocci

Providencia rettgeri - gram -

- rods

- enterobacteriaceae species