sample collection, transportation, and submission of cultures (bacterial and fungal)

what type of agar is blood agar plate?

non-selective

what is the purpose of using a blood-agar plate?

1. can grow fastidious and non-fastidious organisms

2. can visualize variations of hemolysis

what type of agar is macconkey agar plate?

selective and differential

what is the purpose for using a macconkey agar plate?

1. recognition of enterobacteriaceae family (gram negative)

2. differentiate those that ferment lactose from those that do not

what is the appearance of colonies that are lactose fermenting bacteria in a MacConkey agar plate?

red/pink

what is the appearance of non-fermenting lactose colonies in a MacConkey agar plate?

colonies are pale or white and agar will turn yellow

what is the purpose of a Mueller-Hilton Agar?

used in antimicrobial sensitivity testing where a law in required

what is the type of agar for the Mannitol Salt Agar plate?

selective and differential

what is the purpose of using a Mannitol Salt Agar plate?

isolation of stapylococci (coagulase positive)

what inhibits the growth of other bacteria besides staphylococci on the Mannitol Salt Agar?

7.5 % NaCl

what would a red Mannitol Salt agar mean?

alkaline conditions meaning we have Stapyloccocus epidermididis

what would a yellow Mannitol Salt agar mean?

acidic conditions meaning we have Stapylococcus aureus

what type of of agar is Sabouraud Dextrose Agar used for?

selective media for cultivation of yeasts, molds, and aciduric bacteria

what is the purpose of a Sabouraud Dextrose agar?

1. growth and maintenance of both pathogenic and non-pathogenic fungi

2. recovery and total counting of yeasts and molds from environmental samples

what yeasts are typically found on a sabouraud dextrose agar?

candida spp. and aspergillus spp.

what does a negative report on a diagnostic test NOT mean?

does not mean the etiological agent is not present

why might an organism not grow leading to a negative test?

1. bacterium overgrown by contaminants

2. bacterium died during transportation

3. animals stopped shedding bacterium before sample was taken

when must care be exercised to ensure successful culture of causative agents?

1. selection

2. collection

3. shipment of specimens

what are the principles of sample collection?

1. take from affected sites

2. sample early following onset clinical signs

3. take from edge of lesion

4. inform lab whether treatment has started

how do you collect anaerobic bacteria?

1. fine needle aspiration

2. postmortem specimens

3. swabs

what are suitable specimens for anaerobic cultures?

1. normal sterile body fluids

2. surgical specimens from site

3. deep abscess taken aspeptically

4. aspirates from deep wounds

5. blood (aspeptically)

what are NOT suitable specimens for anaerobic culture?

1. saliva

2. vaginal secretions

3. feces (unless specific clostridial pathogen)

4. traceal

5. naso-tracheal aspirates

6. colostomy

7. skin or superficial wound

8. catheterized urine

what are techniques used in staining and microscopy for direct examination of bacteriological species?

1. gram stain

2. dilute carbol fuschin stain

3. ziehl neelsen stain

what are indirect methods used to identify bacteria?

1. ELISA

2. aggulination tests

3. precipitation

4. fluorescent antibody test

what are the advantages of indirect methods of bacterial identification?

1. high output in short amount of time

2. low cost

3. quantitative and qualitative

what is PCR based on?

nucleic acid (DNA/RNA) amplification

what are the advantages to PCR?

1. rapid

2. small amount of sample required

3. highly sensitive and specific

what are the disadvantages to PCR?

1. expensive

2. primers vary in specificity

what are the advantages of molecular methods in bacterial identification?

1. rapid

2. generate easily interperated spectra

3. qualitative and quantitative data

4. relatively low cost

5. high output

what are the disadvantages of molecular methods in bacterial identification?

1. limit for detection of organisms with low abundance

2. host proteins and normal flora may overlap on spectra

3. lack differentiation of closely related species

what media is required to grow Leptospira?

EMJH media

what are the guidelines for bacterial isolation from tissues and organs?

1. use sterile instruments

2. must have 1cm^3 sample collected (anaerobic larger)

3. postmortem material should be collected as soon as possible

4. in case of abortion send whole fetus

what are the guidelines for bacterial isolation from swabs and discharges of selected specimen?

1. fluid preferred to swabs

2. bacteria suscptible to desiccation of collected on dry swab

3. short cotton swabs unsatisfactory for respiratory diseases

4. guarded swabs necessary if contamination of normal flora a possibility

where should fecal sample be obtained from?

directly from rectum in a manner to avoid contamination

why are rectal swabs often inadequate?

not enough fecal matter for agent detection

why do fecal samples need to be collected over several days?

some organisms are shed intermittently

how do we get a bacterial isolation sample from an INDIVIDUAL cow?

1. as soon as masititis is observed

2. collected in sterile vials or tubes

3. clean udders before taking the sample

how do we get bacterial isolation from bulk tank milk (HERD)?

1. presence or absence of bacteria

2. identification of prominent bacterial groups

what is the process for obtaining bacterial isolation from cow? in terms of testing milk

1. udder should not be rinsed with water unless very dirty

2. wipe teat with 70% ethyl alcohol

3. wipe teat FURTHEST away from you, then closer one

4. discard the first milk from each teat

5. for a composite sample, take a little milk from each teat

6. sample milk from NEAREST teats first

what type of sample is taken for bacterial eye specimens?

1. conjunctival swab by gently holding palpebrae apart

2. scaping with fine sterile spatula

how do we collect urine sample?

1. cystocentesis

2. catheter urine

3. midstream urine

what is the ideal sample for abscess?

collect 3 mL of pus with scrapings from the wall of the abscess

what type of bacteria can often be cultured from abscesses?

anaerobic

what type of pus will yield the best sample for an abscess?

pus from freshly formed abscesses

what must we do when taking a sample for skin lesions if there are pustules or vesicles present at the surface?

disinfect with 70% ethyl alcohol

how do you take a skin lesion sample?

aspirate with a sterile syringe and fine needle

where should a swab sample be taken in terms of skin lesion samples?

from the raw surface of the ulcer

what should be collected after the superficial area has been cleaned and debrided?

a biopsy of wound should be taken

how do bacteria grow in liquid media?

binary fission resulting in two daughter cells

what are the four phases of bacterial growth?

1. Lag phase

2. Logarithmic phase (log)

3. Stationary phase

4. Death (decline) phase

what happens in the lag phase of bacterial growth?

size of bacteria increase but the actual number of bacteria remain the same

what happens in the log phase of bacterial growth?

bacterial cells start dividing and number of cells increase exponentially

what happens in the stationary phase of bacterial growth?

the number of cells remain the same due to depletion of nutrients and increase in toxic effects

what happens in the decline phase of bacterial growth?

cell number decreases due to cell death

most bacteria grow...

on cell-free media as colonies

what is the growth of a bacteria in culture influenced by?

1. temperature

2. hydrogen ion concentration

3. availability of moisture

4. atmospheric composition

5. osmotic pressure

what are the types of media based on consistency?

1. broth

2. semisolid

3. solid

what is liquid media available for use in?

1. test-tubes

2. bottles

3. flasks

what are the key characteristics in liquid media of bacteria?

1. bacteria grow uniformly

2. production of turbidity

3. certain aerobic and fimbria from surface pellicle

what does liquid media not contain?

agar

what does nutrient broth grow?

non-fastidious bacteria

what can semi-solid agar demonstrate?

bacterial motility

what does basal media support?

most non-fastidious bacteria

blood agar is an example of what type of media?

enriched media since extra nutrients (blood) are added

selective media

inhibits unwanted commensal or contaminating bacteria

differential media

different bacteria can be recognized on the basis of their colony morphology