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Growing of Bacteria on Sheep and Rabbit Blood Agar

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Materials and methods

Bacterial collection

Growing of bacteria on sheep and rabbit blood agar

For the detection of hemolytic properties the collected field strains and the reference strains of genera Arcanobacterium and Trueperella were comparably cultivated on both sheep and rabbit blood agar. The components of the blood agar (base) (Merck, Darmstadt, Germany)* was as follows:

Nutrient substrate (heart extract and peptones)

20.0 g

Sodium Chloride

5.0 g

Agar

15.0 g

Distilled water

Up to 1000 ml

pH: 6.8 ± 0.2 at 25 ËšC

 

The medium was prepared as instructed by the manufacturer. The components were added to distilled water, gently mixed and heated under repeated agitation, finally boiled for 1 min to completely dissolve the medium then sterilized by autoclaving at 121 ËšC for 15 min. The medium was then cooled to 50 ËšC then 5% of sterile defibrinated sheep or rabbit blood agar using ethylenediaminetetraacetic acid (EDTA, Sarstedt AG & Co., Nümbrecht, Germany) was aseptically added and gently mixed. Sheep and rabbit blood agar were aseptically collected from donor animals of the Institut für Hygiene und Infektionkrankheiten der Tiere and from Zentrales Tierlabor, Justus-Liebig-Universität Gießen, Frankfurterstraße 105, 35392 Gießen, respectively. Of the medium15ml was aseptically dispensed to sterile agar plates and preserved at 4 ËšC preferably in sealed plastic bags (to prevent loss of moisture) ready to be used. The bacterial isolates were then cultivated on sheep and rabbit blood agar plates and incubated at 37 ËšC for 48 to 72 h in an incubator (Infors AG Bottmingen, Switzerland) under microaerobic conditions using a candle jar (Lenz Laborglas GmbH, Wertheim, Germany) and for some isolates under aerobic and anaerobic conditions using the anaerobic system (AnaeroGen™, Oxoid Ltd, Basingstoke, Hampshire, UK) inside anaerobic jar (Anaerocult, Merck). The bacteria were subcultured every four weeks.

______________________________________________________________________________

* If not otherwise stated, all chemicals were obtained from Merck, Darmstadt, Germany.

Determination of smooth and rough colony types

Storage of bacterial cultures

The bacteria were grown on sheep blood agar as described in 3.2. Freshly subcultured bacteria were then collected using a sterile cotton swab (Böttger, Bodenmais, Germany) and delivered into a sterile capped microtube (Sarstedt AG & Co.) containing 1.5 ml of bovine serum (PAA Laboratories GmbH, Cölbe, Germany) with 6% glucose. The tubes were then stored at -80 ËšC.

Identification and further characterization of the bacteria

Determination of hemolysis and CAMP-like hemolytic reactions

Bacterial colony morphology and hemolytic properties were investigated by cultivation of the bacteria on sheep or rabbit blood agar under microaerobic conditions for 48-72 h at 37 ËšC and for some isolates also under aerobic and/or anaerobic conditions (3.2).

The CAMP-like test was originally designed by Christie, Atkins and Munch Peterson in 1944 to identify serological group B Streptococci which produce a CAMP factor causing an enlargement in the zone of hemolysis in the presence of incomplete β-hemolysin from S. aureus. An antagonistic (reverse) CAMP-reaction could be observed in A. haemolyticum and A. phocae (Fraser, 1964; Johnson et al., 2003) and is represented by an arc-shaped zone of inhibition in staphylococcal β-hemolysin zone in the presence of exotoxins of A. haemolyticum and A. phocae. The CAMP-like formation and the antagonistic hemolytic reactions were performed on sheep blood agar (3.2) using β-hemolytic S. aureus, S. agalactiae and R. equi as indicator strains and the reference strains of genera Arcanobacterium and Trueperella (Tab. 1) as controls. The indicator strains were inoculated vertically in the middle of the plate and the strains to be tested and a negative and positive control were streaked in a straight line perpendicular to the indicator strain 2-3 mm far and not touching the streak. The plates were then incubated under microaerobic conditions at 37 ºC for 48 h. A positive CAMP-like reaction appeared as a formation of a half-moon shaped hemolysis as a result of intersection of exotoxin of the investigated strains within the zone of incomplete staphylococcal-β-hemolysis. A positive antagonistic (reverse) hemolytic reaction was observed as an arc-shaped inhibition zone of β-hemolysin of S. aureus.

Synergistic CAMP-like reaction with indicator strains

Positive

Control

Negative

Control

S. aureus (β-hemolysin)

T. pyogenes

DSM 20630áµ€

T. bernardiae

DSM 9152áµ€

S. agalactiae

A. phocae

DSM 10002áµ€

A. pluranimalium

DSM 13483áµ€

R. equi

A. haemolyticum

DSM 20595áµ€

T. bialowiezensis

DSM 17162áµ€

Reverse CAMP-reaction

A. haemolyticum

DSM 20595áµ€

A. phocae

DSM 10002áµ€

T. bialowiezensis

DSM 17162áµ€

Gram-staining and determination of bacterial cell morphology

Gram-staining was carried out as described by Bisping and Amtsberg (1988).

………….

Bacterial motility test

The procedure was achieved using Hitchens motility medium as stab method. A wire loop containing bacterial cultures was stabbed into tubes containing Hitchens semi-solid media (Hitchens, 1922).

The components of Hitchens semi-solid media was as follows:

Meet extract

5.0 g

Peptone

20.0 g

Potassium nitrate

2.0 g

Agar

1.3 g

Aqua dest.

Up to 1000 ml

pH: 7.5 ± 0.2 at 25 ËšC

 

The stabbed tubes were incubated for 48 to 72 h at 37 ËšC under microaerobic conditions using a candle jar. Motility was indicated as a growth of hazy zone or a generalized turbidity around the stabbed line. Non-motile organisms which lack flagella formed a single line of growth that does not spread around.

Controls:

Positive Control

Negative Control

Proteus vulgaris

S. aureus

  • Both controls were kindly obtained from Institut für Hygiene und Infektionskrankheiten der Tiere.

Biochemical characterization of the bacteria

Identification of bacteria using API Coryne Microbial Identification Kit

The API Coryne test was carried out (as instructed by manufacturer) using the API Coryne Microbial Identification Kit (Biomérieux, Nürtingen, Germany). The kit consists of incubation boxes, API Coryne strips and ampules of API GP Medium and API Suspension Medium. Each strip contains 20 labeled microtubules which comprise dehydrated substrates, 11 to detect enzymatic activities (nitrate reduction, pyrazinamidase, pyrrolidonyl arylamidase, alkaline phosphatase, β-glucuronidase, β-galactosidase, α-glucosidase, N-acetyl-β-glucosaminidase, esculin hydrolysis, urease, gelatin hydrolysis and catalase activity) and 8 for carbohydrate fermentation tests (D-glucose, D-ribose, D-xylose, D-mannitol, D-maltose, D-lactose, D-saccharose and glycogen). The incubation box is made up of a tray and a lid. Distilled water was distributed into the honey combed wells of the tray to create a damp climate. API suspension medium was opened (as indicated in the manual) and with a sterile cotton swab (Applimed SA, Châtel-St-Denis, Switzerland) freshly subcultured bacteria were harvested and dissolved into the medium until a suspension of turbidity greater than 6 McFarland was formed. Each microtubule consists of a tube and a cupule. The first 11 microtubules of the strip (NIT to GEL) were filled with approximately 150 μl of the obtained suspension, for URE up to the level of the microtubule and for GEL the whole tube and cupule were covered. The API GP Medium ampule containing phenol red as pH indicator was then opened (as indicated by the manufacturer) and the rest of bacterial suspension transferred into it. The new suspension was thoroughly mixed, then distributed on the rest of microtubules from 0 to GLYG to the level of the microtubule. The cupules of all the underlined microtubules (URE, and 0 to GLYG) were concealed with a layer of mineral oil until a convex surface was formed. The tray was then covered with the lead and incubated for 48 h at 37 ËšC (3.2) under aerobic conditions. After incubation, one drop of Nit 1 and Nit 2 reagent (Biomérieux) were added to nitrate microtubules, respectively, one drop of PYZ reagent (Biomérieux) to PYZ microtubules and 1 drop each of ZYM A and ZYM B reagents (Biomérieux) were added respectively, to the microtubules from (PYRA to β-NAG). All reactions were read by matching colors according to the Reading Table (Biomérieux) and results were recorded on a result sheet. A seven-digit numerical code was obtained and compared with the API Coryne Analytical Profile Index (apiweb TM, version 1.2.1, 2003, Biomérieux) for the identification of the bacteria. Catalase test was performed by adding 1 drop of 3 % H2O2 to the ESC or GEL microtubules. A positive reaction was indicated by bubble appearance.

Determination of bacterial enzymes by fluorescent 4-methylumbelliferyl conjugated substrates

A simple and fast method for the detection of bacterial enzymes, based on the release of the fluorescent reaction product methylumbelliferone, was described by Maddocks and Greenan (1975) and Slifkin and Gil (1983). In the present study the 4-methylumbelliferyl conjugated substrates for detection of β-Glucuronidase, β-Galactosidase, α-Glucosidase, N-acetyl-β-Glucosaminidase (Sigma, Steinheim, Germany) were used as instructed by the above mentioned authors.

A stock solutions containing substrates were prepared by dissolving 15 μmol of β-Glucuronidase, α-Glucosidase, N-Acetyl-β-Glucosaminidase, α-Glucosidase and 5 μmol β-Galactosidase each in 200 μl of dimethyl sulfoxide (Carls Roth GmbH, Karlsruhe, Germany), then each volume was increased to 10 ml by adding 0.2 ml of sodium acetate buffer, pH 5.2.

A loop of freshly cultured bacterial colonies were robustly rubbed onto a filter paper (Macherey-Nagel, Düren, Germany) forming a bacterial smear. Then, 20 μl of 4-methylumbelliferyl-conjugated substrates were placed on the smear and incubated at 37 ËšC for 1 h. After that, 20 μl of 0.1 mol/l NaOH was added to intensify fluorescence, that was investigated by exposing it to UV light (360 nm) in a dark place.

Controls:

Test

Positive Control

Negative Control

β-Glucuronidase

A. pluraniamlium

DSM 13483áµ€

A. haemolyticum

DSM 20595áµ€

β-Galactosidase

A. hippocoleae

DSM 15539áµ€

T. bernardiae

DSM 9152áµ€

α-Glucosidase

T. bonasi

DSM 17163áµ€

A. phocae

DSM 10002áµ€

N-Acetyl-Glucosaminidase

T. pyogenes

DSM 20630áµ€

T. bialowiezensis

DSM 17162áµ€

Determination of α-Mannosidase, α-Glucosidase and β-Glucosidase enzymatic activity

The tests using tablets from Rosco Diagnostica (A/S, Taastrup, Denmark) were performed as instructed by the manufacturer. A dense bacterial suspension (at least McFarland No. 4) of the strain to be tested was prepared in 250 µl of sterile 0.9 % NaCL (Roth) in 1.5 ml Eppendorf tubes (Sarstedt AG & Co.). The solution was vortexed for 30 sec and one diagnostic tablet for the detection of α-Mannosidase, α-Glucosidase and β-Glucosidase, respectively was added. The Eppendorf tubes were then incubated at 37 ºC for 4 h. A yellow colour was an indication for a positive reaction, and a colourless or very faint yellow was considered as a negative reaction.

Controls:

Test

Positive Control

Negative Control

α-Mannosidase

A. pluraniamlium

DSM 13483áµ€

A. hippocoleae

DSM 15539áµ€

α-Glucosidase

A. phocae

DSM 10003

T. bonasi

DSM 17163áµ€

β-Glucosidase

T. abortisuis

DSM 19515áµ€

A. phocae

DSM 10002áµ€

Detection of catalase activity

The enzyme catalase is produced by bacteria that consume oxygen for respiration. It also serves as a protective enzyme that is capable of destroying the toxic product hydrogen peroxide by converting it into water and gaseous oxygen.

2 H2O2 → 2 H2O + O2

Using a sterile loop a few colonies of fresh bacterial cultures were inoculated into 2-3 drops of 3 % H2O2 on a glass slide. The appearance of bubbles was a sign of a positive reaction whereas, a negative reaction was indicated by a uniform turbidity with no bubble formation.

Controls:

Positive Control

Negative Control

A. pluranimalium

DSM 13483áµ€

T. abortisuis

DSM 19515áµ€

Detection of bacterial enzymes using special growth medium

  • Detection of growth on Loeffler medium

Loeffler agar was originally found by Loeffler (1887) to determine microbial proteolytic activities and was later modified by Perry and Petran (1939) and Buck (1949). The medium was composed of a Standard I Nutrient Broth with bovine serum.

Standard I Nutrient Broth ingredients:

Peptone

15.0 g

Yeast extract

3.0 g

Sodium Chloride

6.0 g

Dextrose

1.0g

Aqua dest.

up to 1000 ml

pH: 7.2 ± 0.2 at 25 ËšC

 

Loeffler medium was prepared by adding 7 : 3 of sterile bovine serum (PAA Laboratories GmbH) and Standard I Nutrient Broth (containing 6 g of glucose per liter), respectively. The mixture was stirred thoroughly then dispensed into small petri dishes. The medium was then left to coagulate in a humid chamber at 90 ËšC for about 50 min and stored at 4 ËšC ready to be used.

A line of bacterial colonies was streaked on the loeffler medium and incubated for 48 h at 37 ËšC under microaerobic conditions (3.2). The streaked line was observed for a groove formation underneath as a sign of proteolytic activities indicating a positive reaction (Hartwigk and Marcus, 1962).

Controls:

Positive Control

Negative Control

T. pyogenes

DSM 20630áµ€

T. abortisuis

DSM 19515áµ€

Loeffler, F. 1887. Darauf theilte Herr Loeffler en einem Zweiten vortrag die ergebnisse seiner weiteren unter suchungen uber die Diphtherie- Bacillen mit. Zentralbl. Bakteriol. Parasitenkd. 2:105-112.

Perry, C.A., and E.I. Petran. 1939. Routine laboratory examinations for C. diphtheriae. J. Lab. Clin. Med. 25:71-78.

Buck, T.C. 1949. A modified Loeffler's medium for cultivating Corynebacterium diphtheriae. J. Lab. Clin. Med. 34:582-583.

  • Detection of Caseinase enzyme activity

Casein consists of a mixture of phosphoproteins present in milk in the form of water-soluble calcium salt of a phosphoprotein. Caseinase enzymes are exoenzyme produced by some bacteria to degrade the milk protein casein by hydrolyzing it into smaller chains of aminoacids and peptides. The test was carried out by cultivating the bacteria on casein agar also known as skim milk agar. The hydrolysis reaction causes the opaque milk agar to turn into a clear zone around the growth area.

Casein agar ingredients:

The bacterial cultures were inoculated as a streak onto the Casein agar plates then incubated at 37 ºC for 48 h under microaerobic conditions (3.2). After incubation, 10 % of trichloroacetic acid (Sigma-Aldrich Chemie GmbH, Munich, Germany) was poured into the plates.


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