Equipment

Milk samples from each quarter are collected in a clean CMT Paddle. The CMT paddle has four shallow cups marked A, B, C, and D to help identify the individual quarter from which the milk was obtained. The CMT solution should be reconstituted according to package instructions.

Procedure: 

1. After discarding the first three to four squirts of foremilk, Take about 2 cc of milk from each quarter. This is the amount of milk that would be left in the cups if the CMT Paddle were held nearly vertical
2. Add an equal amount of CMT solution to each cup in the paddle.
3. Rotate the CMT Paddle in a circular motion to thoroughly mix the contents. Do not mix more than 10 seconds.
4. Read the test quickly. Visible reaction disintegrates after about 20 seconds. The reaction is scored visually. The more gel formation, the higher the score.

Interpretation:

Youtube link: CMT: A simple and useful tool for Subclinical mastitis (https://www.youtube.com/watch?v=YRbH_E7JtTU)

Reference: 

1. Council  NM. 2017. Laboratory handbook on bovine mastitis. NMC Inc., Madison.

Somatic cell count (SCC) by DeLaval cell counter (DCC)

Bulk milk sample: 

• Ensure that the bulk tank milk sample is taken after the sample is agitated for five minutes 
• Use a clean sanitized dipper and a clean vial or 50 ml falcon tube
• Take the sample from the centre of the bulk tank. Never take the sample from the top.

 

Storing the milk: 

• Fresh and preserved milk sample can be kept at refrigerated temperature for up to two days before measurement. 
• Milk samples that have been frozen do not give reliable results

 

Somatic Cell Measurement;

• Take a cassette out of the Aluminum bag (don’t touch cassette measuring window)
• Mix the sample gently by turning the vial over a few times. The temperature of the sample should be between 10-40° C (Don’t shake the milk sample vial before analysis. It can affect the cell count)
• Place the inlet of the cassette in the milk sample vial
• Press the piston
• Check that the sample has been loaded to half of the lane 3
• The milk sample can be kept in the cassette for up to 2 minutes before measurement with reliable result. 
• Place the cassette in the DCC holding the cassette inlet to the left.
• Close the sample insertion lid. 

Interpretation:

BTSCC/ml	Infected quarters in herd (%)	Production loss (%)
200,000	6	3
500,000	16	6
1,000,000	32	18
1,500,000	48	29

 

Reference: 

1. DeLaval Cell Counter DCC) manual (2005)
2. Council  NM. 2017. Laboratory handbook on bovine mastitis. NMC Inc., Madison.

Link: https://www.youtube.com/watch?v=w_UrQVTofDM

Antibiotic Susceptibility Test

Indication:

The test determines the susceptibility of a microbial species against different antibiotic agents.

Factors affecting antibiotic susceptibility testing

pH:

pH of the medium is an important factor which influences the accuracy of the antibiotic susceptibility testing. If the pH of the medium is too low than the desired pH, certain drugs such as amino glycosides, quinolones and macrolides  lose their potency, on the other hand, antibiotic classes such as tetracyclines appear to have excess activity a lower Ph and the vice versa happens in the case of the higher pH.

Moisture:

The presence of moisture content on the medium can counter act with accuracy of the susceptibility testing. It is important to remove the excess moisture present in the agar surface, by keeping it in the laminar flow hood for few minutes.

Effects of medium components:

If the media selected for the antibiotic susceptibility contains excessive amounts of thymine or thymidine compounds, they will reversibly inhibit the action of certain antimicrobial agents such as trimethoprim groups. This reversible inhibition yields smaller or less distinct or even no zones and will be misinterpreted as resistant antibiotics. MHA is low in thymine and thymidine content and it can be used successfully to study the susceptibility of antibiotics. Also the medium containing excessive cation reduces the zone size, while low cation content results in unacceptably large inhibition zones.

Amount of organism:

The amount of the organism used for the susceptibility testing is standardized using a turbidity standard. This is obtained by a visual approximation using McFarland standard of 0.5 or else it can be determined by using a spectrophotometer with Optical density of 1 at 600 nm wavelength.

Materials Required:

1. Petri-disc containing microbial culture (For example, Escherichia coli) or Samples (Liver, spleen)
2.  Inoculation loop
3. Bunsen burner
4. Phosphate Buffer Saline (PBS) solution
5. McFarland solution
6. Mueller-Hinton agar (MHA) plate or a nutrient agar (NA) plate 
7. Cotton swab
8. Antibiotic disks (Streptomycin(S), Ciprofloxacin(CIP), Chloramphenicol(C), Doxycycline (D), Penicillin G (P), Gentamycin (G)
9. Tooth pick
10. Incubator
11. Ruler

Procedure:

1. Take the samples (Liver, spleen, milk) or pure stock of bacteria 
2. Inoculate on Blood agar, MHA or NA
3. Select a pure culture plate of one of the organisms to be tested.

1. Aseptically emulsify a colony from the plate in the 5ml sterile Phosphate Buffer Saline (PBS) solution. Mix it thoroughly to ensure that no solid material from the colony is visible in the saline solution.

1. Repeat until the turbidity of the saline solution visually match that of the standard turbidity (0.5% McFarland turbidity)
2. Take a sterile swab and dip it into the broth culture of organism.
3. Gently squeeze the swab against the inside of the tube in order to remove excess fluid in the swab.
4. Take a sterile Mueller-Hinton agar (MHA) plate or a nutrient agar (NA) plate.
5. Use the swab with the test organism to streak a MHA plate or a NA plate for a lawn of growth.
6. After the streaking is complete, allow the plate to dry for 5 minutes.
7. Antibiotic discs can be placed on the surface of the agar using sterilized forceps.
8. Gently press the discs onto the surface of the agar using flame sterilized forceps or inoculation loop.
9. Carefully invert the inoculated plates and incubate for 16-24 hours at 37° C.
10. After incubation, use a metric ruler to measure the diameter of the zone of inhibition for each antibiotic used.
11. Compare the measurement obtained from the individual antibiotics with the standard table to determine the sensitivity zone.
12. Compare the measurement obtained from the individual antibiotics to the standard table to determine whether the tested bacterial species is sensitive or resistant to the tested antibiotic.

Interpretation:

Susceptible or Intermediate or Resistant  

Standard chart for measuring diameter of zone of sensitivity

Preparation of 0.5% turbidity:

1. 1% BaCl2- Mix 1.17 gm BaCl2 in 99 ml water.
2. 1% H2SO4- Mix 1 ml H2SO4 in 99 ml water.
3. Discard 0.5% ml from 1% H2SO4 solution and mix 0.5 ml from 1% BaCl2 solution.

References:

• Cappuccino G .James, Sherman Natalie, Microbiology A laboratory manual, seventh edition, Pearson Education

• Amrls.cvm.msu.edu/microbiology/detecting-antimicrobial-resistance/test-methods/examples-of-antibiotic-sensitivity-tesing-methods

• Link: https://www.youtube.com/watch?v=BXr_kcki4Ag

 

Lab information:

1. Poultry Research and Training Centre (PRTC), CVASU, Zakir Hossain Road, Khulshi, Chattogram-4225, Phone: +88-031-2566372, email: [email protected]
2. Medicine lab, Dept. of Medicine and Surgery, CVASU, Zakir Hossain Road, Khulshi, Chattogram-4225
3. Microbiology lab, Dept of Microbiology and Veterinary Public Health, CVASU, Zakir Hossain Road, Khulshi, Chattogram-4225
4. Animal Nutrition lab, Dept of Animal Science and Nutrition, CVASU, Zakir Hossain Road, Khulshi, Chattogram-4225
5. Udder Health Bangladesh Laboratory, SAQTVH building (Second floor), CVASU, Khulshi, Chattogram

Isolation and identification of bacteria

• Staphylococcus aureus (SA): 

Criteria	Staphylococcus aureus (SA)
Cultural properties
Blood agar (BA)	Golden yellow colony with beta hemolysis
Mannitol Salt Agar (MSA)	Yellow colonies with yellow zones
Gram staining reaction	gram positive cocci arranged in grape like clusters and motile
Biochemical test
Catalase test	Positive indicated by bubble production
Coagulase test	Positive indicated by the clumping of the organisms
Sugar fermentation	Produces acid without gas from dextrose, lactose, maltose, mannitol and sucrose which is indicated by yellow color in the MSA
Motility test	Non-motile
KOH test	Negative indicated by no formation of sticky slime

 

• Coagulase-negative staphylococci (CNS):

Criteria	Coagulase-negative staphylococci (CNS)
Cultural properties
Blood agar (BA)	Moderately-sized and white or golden color colonies and non-hemolytic
Mannitol Salt Agar (MSA)	Small red colonies with no color change to the medium
Gram staining reaction	Gram positive cocci arranged in grape like clusters and motile
Biochemical test
Catalase test	Positive indicated by bubble production
Coagulase test	Negative
Motility test	Non-motile
KOH test	Negative indicated by no formation of sticky slime

 

• Streptococcus sp. :

Criteria	Streptococcus sp.
Cultural properties
Blood agar (BA)	Alpha or beta hemolytic small translucent dew drop like colonies
Gram staining reaction	Gram-positive cocci and characteristic chains forming
Biochemical test
Catalase test	Negative
KOH test	Negative indicated by no formation of sticky slime

 

• Bacillus sp.: 

Criteria	Bacillus sp.
Cultural properties
Blood agar (BA)	Bacillus cereus colonies are large, slightly gray with irregular edges, and often surrounded by a clear zone of hemolysis after 24 to 48 hours  Bacillus subtilis colonies are greenish-gray with a ground glass appearance
Nutrient agar (NA)	Thick creamy color colony
Gram staining reaction	Large, gram-positive rods, cells occur singly, in pairs or in long chains
Biochemical test
Catalase test	Positive indicated by bubble production
Oxidase test	Negative
Motility test	Motile
KOH test	Negative indicated by no formation of sticky slime

 

• Escherichia coli (E. coli):

Criteria	Escherichia coli (E. coli)
Cultural properties
Blood agar	Gray, moist, opaque and have a fecal odor, generally non-hemolytic
MacConkey Agar (MAC)	Large, pink to red, dry colony surrounded by pink zone
Eosin methylene blue (EMB)	Greenish metallic sheen
Gram staining reaction	Gram-negative, pink color, motile rod shape arranged in single or pair
Biochemical test
Indole test	Positive indicated by formation of pink to red color in the reagent layer on the top of test tube
Motility	>90% are motile
MR test	Positive indicated by red color at the  surface of the medium as a result of high acid production
VP test	Negative indicated by yellow color at the surface of the medium
KOH test	Positive by formation of sticky slime
Sugar fermentation test	Produces acid and gas from dextrose, lactose, maltose, mannitol and sucrose which is indicated by yellow
Oxidase test	Negative indicated by no color change

• Enterobacter sp. :

Criteria	Enterobacter sp.
Cultural properties
Blood agar	Gray, moist  and have a fecal odor, generally non-hemolytic
MacConkey Agar (MAC)	Large, mucoid colonies and surrounding medium was pink due to acid production from lactose
Eosin methylene blue (EMB)	Large, mucoid, blue-black
Gram staining reaction	Gram-negative, pink color, motile rod shape arranged in single or pair
Biochemical test
Indole test	Negative indicated by formation of yellow color in the reagent layer on the top of medium
Motility	Motile
MR test	Negative indicated by yellow color at the  surface of the medium
VP test	Positive indicated by red to pink color at the surface of the medium
KOH test	Positive by formation of sticky slime
Simmons citrate utilization test	Positive indicated by blue color medium from green color
Sugar fermentation test	Produces acid and gas from dextrose, lactose, maltose, mannitol and sucrose which is indicated by yellow
Oxidase test	Negative indicated by no color change

 

• Klebsiella sp. :

Criteria	Klebsiella sp.
Cultural properties
Blood agar	Gray, moist to mucoid, generally non-hemolytic
MacConkey Agar (MAC)	Pale pink to yellow mucoid colonies due to capsule
Eosin methylene blue (EMB)	Large, mucoid, pink to purple colonies with no metallic green sheen
Gram staining reaction	Gram-negative, pink color, motile rod shape arranged in single or pair
Biochemical test
Indole test	Negative indicated by formation of yellow color in the reagent layer on the top of medium
Motility	Non-motile
MR test	Negative indicated by yellow color at the  surface of the medium
VP test	Positive indicated by red to pink color at the surface of the medium
KOH test	Positive by formation of sticky slime
Simmons citrate utilization test	Positive indicated by blue color medium from green color
Sugar fermentation test	Produces acid and gas from dextrose, lactose, maltose, mannitol and sucrose which is indicated by yellow
Oxidase test	Negative indicated by no color change

 

1. Pseudomonas sp. :

Criteria	Pseudomonas sp.
Cultural properties
Blood agar	White to gray color coloy with irregular edges, usually hemolytic, grape like odor
MacConkey Agar (MAC)	Translucent, shiny coloy
Eosin methylene blue (EMB)	Irregular colorless or pinkish mucoid colonies
Gram staining reaction	Gram-negative, pink color, motile rod shape arranged in single or pair
Biochemical test
Indole test	Negative indicated by formation of yellow color in the reagent layer on the top of medium
Motility	Motile
MR test	Negative indicated by yellow color at the  surface of the medium
VP test	Negative indicated by yellow color at the  surface of the medium
KOH test	Positive by formation of sticky slime
Simmons citrate utilization test	Positive indicated by blue color medium from green color
Sugar fermentation test	Negative
Oxidase test	Positive indicated by color change (Blue)
Catalase test	Positive indicated by bubble formation

Maintenance of stock culture:

1. Fifty percent sterile buffered glycerin was made by mixing 50 parts of pure glycerin and 50 parts of buffered glycerin saline 
2. Pure culture of isolated bacteria from blood agar was incubated overnight in Brain heart infusion broth 
3. Then the bacteria were mixed with 50% sterile buffered glycerin in 1.5 ml eppendrof tubes (700µl broth culture and 300µl gluserol) 
4. Then preserved at -80o C for long term use

Procedure of biochemical tests

Catalase test:

Small amount of colony was placed on a fresh, clean and grease free slide. One drop of 3% H2O2 poured onto the colony, a cover slip was placed and bubble formation was indicated as positive result.

Coagulase test:

Horse plasma collection: Whole blood from horse was collected into commercially available sterile tubes containing EDTA to perform the test. Then blood was centrifuged at 2600 rpm for 10 minutes using a refrigerated centrifuge device. The resulting supernatant, the plasma, was then immediately transferred to a sterile 1.5 ml eppendorf tube using sterile tips and stored at -20ºC for future use.

Tube coagulase test: From each tube cultivated in BHIB, 50 µL was transferred to sterile tubes containing 50 µL of horse plasma. The incubation was done at a temperature of 37ºC for 6 hours. The presence of coagulates was justified, considering large organized coagulation and coagulation of all the contents of the tube which do not come off when inverted. A control tube also is placed to validate the result.

Indole test:

Pure bacterial culture was grown in sterile peptone broth/ Brain Heart Infusion Broth (BHIB) for 24 hours. Following incubation, 4-5 drops of Kovac’s reagent was added to the culture broth. A positive result was reflected by the presence of a red or red-violet color in the surface layer of the broth. A negative result appears yellow. A variable result can also occur, showing an orange color as a result.

Oxidase test:

A piece of filter paper was placed in a clean petri dish and 2 drops of oxidase reagent was added to filter paper. A colony of test organism was removed using a wire loop (not an oxidized wire loop) and rubbed onto treated filter paper. A color change to blue/ dark purple within 5 to 10 seconds was considered as positive test. Microorganisms were oxidase negative if the color did not change.

Methyl red (MR) test:

Using a light inoculum, tubes of MR-VP media were inoculated with 24-hour pure cultures of test organisms. Then tubes were incubated aerobically at 37°C for a minimum of 48 hours. After incubation, 2.5 ml of culture was transferred into a new sterile culture tube and 5 drops of the methyl red reagent was added to the tube. Red color at surface of the medium as a result of high acid production and a decrease in the pH of the culture medium was indicated as positive test. Yellow color at surface of the medium was indicated negative test.

Voges-Proskauer (VP) test:

0.6 ml (or 12 drops) of Barritt's reagent A was added to the remaining 2.5 nil (after MR test) of culture grown in MR-VP broth. Then 0.2 ml (or 4 drops) Of Barrit’s reagent B was added and carefully shaken the tube for 30 seconds to expose the medium to atmospheric oxygen. Then the tube was allowed to stand for at least 30 minutes. Red coloration on top of the culture was considered as VP positive.  Yellowish color at surface of the medium was considered as VP positive.

Sugar fermentation test:

Each tube containing peptone water sugar broth was inoculated with a small amount of test organism and incubated at 37°C for 24 hours. Fermentation was indicated by a yellow color in the medium due to acid production. Gas production was indicated by the presence of gas bubbles in the fermentation tube.

Composition of different reagents:

Kovac’s reagent
Para-dimethylaminobenzaldehyde (DMAB)	2g
Isoamyl alcohol	30ml
HCL (Concentrated)	10ml
Methyl Red solution
Methyl red
Ethanol (95%)	60ml
Distilled water	40ml
Voges-Proskauer reagents
Barritt’s reagent A (VP-1)
Aloha-naphthol	5g
Ethanol (95%)	100ml
Barritt’s reagent B (VP-2)	0.02g
NaOH	40g
Distilled water	100ml
Oxidase reagent
Tetra-methyl-p-phrnylenediamine dihydrochlorid	1g
Distilled water	100ml

 

References:

1. Council  NM. 2017. Laboratory handbook on bovine mastitis. NMC Inc., Madison.
2. Lennette EH. Balows  A. Hausler Jr WJ. Shadomy HJ. 1985. Manual of clinical microbiology. American Society for Microbiology, Washington, DC.