Basic Swine Bacteriology

Enteric Pathogens
Escherichia coli
Blood agar Most pathogenic strains will be β haemolytic or smooth and mucoid	MacConkey’s Pink colonies – lactose fermenter	Tergitol Yellow- gold colonies lactose fermenter
A B C	Tests – A – Urease –ve yellow B – Simm’s + ve red colour C – Kligler’s +ve with gas Oxidase -ve	Gram negative rod
Clostridium perfringens
	Requires anaerobic culture. Note flat spreading colony shape. Double zone of haemolysis.		Gram positive rods – vegetative and solid rods some spore formation
Blood agar
Brachyspira hyodysenteriae and Brachyspira pilosicoli
	Requires anaerobic culture and 42C. A filmy surface growth is typical, rather than distinct individual surface colonies. β haemolytic Bacteria lie under the surface.			Weakly Gram positive spirochaetes.

Salmonella spp. There are over 2300 types of salmonella
Blood agar Salmonella strains are non-haemolytic on blood agar	MacConkey’s A non-lactose fermenter		Tergitol – a non-lactose fermenter
A B	Tests – A – Kligler’s Iron agar –H2S + Lactose Blue colour B - Lysine Iron Agar + ve Blue colour	Gram negative rod
Lawsonia intracellularis
Note L. intracellularis requires cell culture. It will not grow on agar media

Respiratory pathogens
Actinobacillus pleuropneumoniae
Blood agar Requires staphylococcus streak - Haemolytic - satellitism		MacConkey’s – No growth					Tergitol - No growth
APP left, HPS right plates		Differentiation between APP and HPS by cultural characteristics and urease positive – blue coloration and is CAMP positive.					Haemolytic colonies
			Gram negative rods and coccobacillary forms Organism generally requires NAD (Nicotinamide adenine dinucleotide to grow – V factor) – supplied by a Staphylococcus streak.
Bordetella bronchiseptica
Blood agar - Incubate for at least 48 hours		MacConkey’s - non lactose fermenter					Tergitol – non lactose fermenter
A B C D			A – Kligler’s –ve red B – Simm’s – Poor reaction little growth C- urease – Strong positive – pink slant D - Simmon’s citrate agar – positive – blue color Oxidase +ve		Small gram negative coccobacilli
Pasteurella multocida
Blood agar assisted with Staphylococcus streak to distinguish A and D by producing mucoid colonies				MacConkey’s – no growth Tergitol – no growth
P. multocida A mucoid colony - may run together	P. multocida D non mucoid – dry colony
A B C			Tests – note distinctive odor – musky A – Dextrose Broth - +ve red no gas. B – Lactose –ve yellow C - Indole positive red In addition - urease negative			Small Gram negative rod or coccobacillus
Salmonella choleraesuis See enteric pathogens for growth characteristics
Streptococcus suis
Blood agar		MacConkey’s – no growth					Tergitol – no growth
		Note type of haemolysis α haemolysis - green, β haemolysis – clearing Catalase –ve					Gram positive small cocci –in chains or pairs

Actinobacillus suis
Blood agar – wide zone of β haemolysis	MacConkey’s – no growth Sometimes very small				Tergitol – no growth
A B C	Tests – No growth on MacConkey’s or Tergitol important differential from E. coli A - Kligler’s – Lactose +ve Dextrose +ve red B – Simm’s – difficult little reaction C – Urease +ve red				Gram negative rod
Haemophilus parasuis
Blood agar – very small colonies around the Staphylococcus streak			MacConkey’s – no growth			Tergitol – no growth
		Can be difficult to grow – requires NAD. Reputedly Grows best under CO2 on chocolate agar. It is urease negative - yellow – useful to distinguish from APP positive - blue.		Gram negative coccobacillus

Arcanobacterium pyogenes
Blood agar Catalase -ve	MacConkey’s – no growth	Tergitol – no growth
	Gram positive pleomorphic rods – “Chinese letters”
Mycoplasma hyopneumoniae
Mycoplasma’s require special media. M. hyopneumoniae requires a very long incubation period and a specialized laboratory to grow the organism, which even then is frequently overgrown with other mycoplasma spp.

Other pathogens
Erysipelothrix rhusiopathiae
Blood agar – small colonies	MacConkey’s – no growth			Tergitol – no growth
		Tests - Produces hydrogen sulfide (black streak) along stab line in Kligler’s iron agar.	Slender Gram positive rod
Actinobaculum suis (Eubacterium suis)
Gram positive pleomorphic rods
				Tests – urease positive
Blood agar – requires anaerobic culture. Flat colonies fried egg shaped	Pleomorphic rods
Staphylococcus spp. – Example - Staphylococcus hyicus
Blood agar	MacConkey’s No growth			Tergitol No growth
	Gram positive cocci – in clumps resembling grapes Catalase positive

Basic antimicrobial tests
Kirby-Bauer
		Note each disc has its own zone of inhibition
Minimum Inhibition Concentration - MIC
Automated system	View of the plate – demonstrating growth (white areas) and inhibition (clear)
	E test MIC strip. The zone of clearing visibly seen on the plate.

Specific Tests:

Gram’s Stain:

Always place organisms in centre and always use “frosted side up”

1. Make a thin smear of the organism with a tiny droplet of water on a slide
2. Dry – best if air dried
3. Fix by gently hearing 2-3 times through the flame
4. Flood smear with Crystal violet for 5 seconds, hold side perfectly level, then dump
5. Wash with distilled water
6. Flood smear, with Gram’s iodine for 5 seconds
7. Wash with distilled water
8. Decolorise with acetone-alcohol about 3 seconds
9. Wash with distilled water
10. Counterstain with safranin for 5 seconds
11. Wash with distilled water
12. Blot slide dry, face down on a paper towel.

Acid Fast: Zeil-Neilson

1. Make a thin smear of the organism with a tiny droplet of water on a slide
2. Fix by gently hearing 2-3 times through the flame
3. Flood smear with Carbol fuchsin
4. Heat flooded slide over flame for 2-3 minutes
5. Wash with distilled water
6. Decolorise with acetone-alcohol about 3 seconds
7. Wash with distilled water
8. Counterstain with methylene blue for 30 seconds
9. Wash will distilled water
10. Blot slide dry, face down on a paper towel.

Catalase Test:

1. Dip a capillary tube into 3% H₂0₂

2. Touch a colony

3. Observe the tube for bubble indicating a positive reaction

Do not contaminate the bacterial colony with blood agar as red blood cells contain catalase thus resulting in a false result. Old cultures can lose their catalase activity.

Oxidase Test:

1. Hold a piece of oxidase test paper with forceps and touch onto an area of heavy growth

2. Rapid (within 10 seconds) purple positive. If within a minute delayed positive

Note an oxidase organism will also be catalase positive

Basic Swine Bacteriology – Summary Table

Organism

Gram Stain

Growth

Sugars and reactions

Anaerobe only

Hamolytic Blood agar

MacConkey

Tergitol

Catalyse

Oxidase

Dextrose broth

Kliglers

Kliglers iron

Lactose

Lysine

Simms

Simms Citrate

Urease

Actinobaculum suis

+B

+

N

-

-

-

-

+

Actinobacillus pleuropnumoniae

-CB

Y

-

-

V

V

+

Actinobacillus suis

-B

β

-

-

+

V

+

+

P

+

Arcanobacterium pyogenes

+B

N

-

-

-

-

Bordetella bronchiseptica

-CB

N

+NL

+NL

+

+

-

P

+

Brachyspira

+S

+

β

-

-

-

-

Clostridium perfringens

+B

+

Y

-

-

-

-

Erysipelothrix rhusiopathiae

+B

N

-

-

-

-

+

Escherichia coli

-B

N+β

+L

+L

+

-

+G

+

-

Haemophilus parasuis

-CB

N

-

-

+

-

-

Pasteurella multocida

-CB

N

-

-

+

+

+

-

+

-

Salmonella

-B

N

+NL

+NL

+

-

+

+

Staphylococcus

+C

-

-

+

-

Streptococci

+C

α/ β

-

-

-

-

Code:

+ and green = Positive. – and red = Negative.

Gram stain: B = Bacillus/coccobacillus. C = Coccoid. CB = Coccobacillus. S = Spirochaete.

Sugars and reactions: V= variable G = Gas. P = Poor L = Lactose NL= Non Lactose

Haemolytic Y = Yes and type α or β. N= No.

Gram’s staining of the main organisms