Udder Health Systems

Specialty Agars

Udder Health Systems, Inc.

Fax 208-343-2046E-mail media@udderhealth.com

4455 S. Meridian Rd.Meridian, ID 83642Voice 877- 398-1360

Udder Health Systems, Inc. FTP/Agar/ Agars Price List 2011

Udder Health Systems, Inc.Available Agars:

Agar Pricing1-99 plates 100-399 plates 400+ platesCamp Agar

Inulin AgarMP2 Agar

MacConkey AgarModified Edwards Agar

Salt AgarWashed Cow Blood Agar

$1.45/plate $1.20/plate $1.00/plate

Mycoplasma AgarPrototheca Agar $2.45/plate $2.25/plate $2.00/plate

TNT Plates $2.45/plate $2.25/plate $2.00/plate*The above agars may be combined in any quantity to achieve the price breaks.

Testing Supplies

Tube Bottle100ml 200ml 500ml

Bovine PlasmaCoagulase Test (1 ml tube) $1.00 $40 $60 $120

Mycoplasma Broth (3 ml tube) $1.00 ---NA--

877-398-1360 Rev: 05/06/2011www.udderhealth.com

Quality Control Organisms:

Udder Health Systems is offering these organisms for Quality Control.Organisms are shipped out on agar plates Next Day Fed Ex.

OrganismStrep. agalactiae

Staph. aureusMycoplasma bovis

Strep. uberisStrep. dysgalctia

Staph. sppE. coli

Klebsiella pneumoniaePseudomonas spp

Pseudomonas aeruginosaArcanobacterium pyogenes

Bacillus spp

Pricing

Quantity PriceSingle QC Organism $25 plus shipping

Udder Health Systems, Inc. FTP\Agar\Blood Agar.doc877-398-1360 Rev: 5/1/2007

Udder Health SystemsWashed Cow Blood Agar

PurposeThis is the standard isolation agarused at Udder Health SystemsLaboratory for cultivating a widerange of mastitis organisms fromindividual cow and bulk tank milksamples.

DescriptionThis cherry red, translucent agar is prepared from a blood agar base with theaddition of 5% washed bovine erythrocytes and ½ strength esculin. This bloodagar has been formulated to enhance the detection of Staphylococcus aureus Alphaand Beta hemolysin when present. The addition of esculin enhances the capabilityof differentiating Streptococci. Certain Streptococci will hydrolyze esculin,causing a mild background blackening for E-strep and a mild greening or nobackground coloration for suspect Streptococcus agalactiae and Streptococcusdysgalactiae organisms. All suspect Streptococcus agalactiae organisms would bemoved to our special Camp media. UHS blood agar will support good growth of awide variety of fastidious microorganisms. When reading this agar, anticipateesculin hydrolysis on some coliforms as well.

Refer to table on back for expected culture responses.

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Udder Health SystemsWashed Cow Blood Agar

Culture Response

Organism Growth ReactionStrep. agalactiae Excellent Esculin negativeStaph. aureus Excellent Hemolysin detectionMycoplasma Not ApplicableStrep. dysgalactiae Excellent Esculin negativeStrep. uberis Excellent Esculin positiveE- strep Excellent Esculin positiveStaph. species ExcellentE. Coli ExcellentKlebsiella pneumoniae ExcellentKlebsiella species ExcellentPseudomonas species ExcellentPseudomonas aeruginosa ExcellentPasteurella ExcellentProteus ExcellentSerratia ExcellentBacillus ExcellentYeast ExcellentMold ExcellentNocardia ExcellentPrototheca ExcellentArcanobacterium pyogenes ExcellentC. bovis Excellent

Udder Health Systems, Inc. FTP\Agar\Modified Edwards agar.doc877-398-1360 Rev: 5/1/2007

Udder Health SystemsModified Edwards Agar

PurposeThis is a special isolation agar used atUdder Health Systems Laboratory forselection of Streptococcus agalactiaefrom individual cow and bulk tank milksamples.

DescriptionThis dark purplish red, translucent agar is prepared from a blood agar base with theaddition of 5% washed bovine erythrocytes and Beta hemolysin. This agar hasbeen formulated with inhibitors to discourage a variety of non-target organisms.The addition of Beta hemolysin allows for the capability of differentiatingStreptococci by providing a camp-like hemolytic reaction, typically within 24hours. The media will exhibit a complete circular hemolysis around each suspectStreptococcus agalactiae colony. All suspect Streptococcus agalactiae organismsshould be moved to our special Camp media for confirmation. The inhibitors inthe media will inhibit Staphylococcus and most coliform organisms.

Refer to table on back for expected culture responses.

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Udder Health SystemsModified Edwards Agar

Culture Response

Organism Growth ReactionStrep. agalactiae Excellent Complete HemolysisStaph. aureus InhibitedMycoplasma Not ApplicableStrep. dysgalactiae Excellent Hemolytic negativeStrep. uberis Excellent Hemolytic variableE- strep Excellent Hemolytic negativeStaph. species InhibitedE. Coli InhibitedKlebsiella pneumoniae InhibitedKlebsiella species InhibitedPseudomonas species FairPseudomonas aeruginosa WeakPasteurella InhibitedProteus InhibitedSerratia InhibitedBacillus InhibitedYeast InhibitedMold InhibitedNocardia InhibitedPrototheca InhibitedArcanobacterium pyogenes InhibitedC. bovis Inhibited

Udder Health Systems, Inc. FTP\Agar\Salt agar.doc877-398-1360 Rev: 5/1/2007

Udder Health SystemsSalt Agar

PurposeThis selective agar is used at UdderHealth Systems Laboratory for selectionof all Staphylococci from bulk tank milksamples.

DescriptionThis crimson red, translucent agar is prepared from a Mannitol Salt agar base withthe addition of 5% washed bovine erythrocytes. This agar has been formulated toenhance the detection of Staphylococcus aureus when present. The addition ofblood to the agar provides the capability of differentiating Staphylcocci usinghemolysin patterns. All suspect Staphylococcus aureus suspects should becoagulase tested. UHS Salt agar will support good growth of Staphylococci, whilethe salt content will inhibit most other bacteria.

Refer to table on back for expected culture responses.

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Udder Health SystemsSalt Agar

Culture Response

Organism Growth ReactionStrep. agalactiae InhibitedStaph. aureus Excellent Yellow

Hemolysin variableMycoplasma Not ApplicableStrep. dysgalactiae InhibitedStrep. uberis InhibitedE- strep Weak ClearStaph. species Excellent YellowE. Coli InhibitedKlebsiella pneumoniae InhibitedKlebsiella species InhibitedPseudomonas species InhibitedPseudomonas aeruginosa InhibitedPasteurella InhibitedProteus InhibitedSerratia InhibitedBacillus InhibitedYeast InhibitedMold InhibitedNocardia InhibitedPrototheca InhibitedArcanobacterium pyogenes InhibitedC. bovis Inhibited

Udder Health Systems, Inc. FTP\Agar\Camp agar.doc877-398-1360 Rev: 5/1/2007

Udder Health SystemsCamp Agar

PurposeThis is a specialized indicator agarused at Udder Health SystemsLaboratory for confirmation ofStreptococcus agalactiae suspects.

DescriptionThis cherry red, translucent agar is prepared from a blood agar base with theaddition of 5% washed bovine erythrocytes and full strength esculin. This bloodagar has been formulated to enhance the presentation of the Beta hemolysin zoneof Staphylococcus aureus. The addition of esculin helps to distinguish Camppositive Streptococccus uberis from Streptococcus agalactiae. Certain Streptococciwill hydrolyze esculin, causing a background blackening for E-strep and a mildgreening or no background coloration for Streptococcus agalactiae andStreptococcus dysgalactiae organisms. Streptococcus agalactiae growth within theBeta hemolysin zone will exhibit the characteristic wedge shaped completehemolysis of the CAMP reaction. All Camp positive, esculin negative organismsare recorded as Streptococcus agalactiae.

Refer to table on back for expected culture responses.

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Udder Health SystemsCamp Agar

Culture Response

Organism Growth ReactionStrep. agalactiae Excellent Camp Positive

Esculin negativeStaph. aureus Excellent Hemolysin detectionMycoplasma Not

ApplicableStrep. dysgalactiae Excellent Camp negative

Esculin negativeStrep. uberis Excellent Camp variable

Esculin positiveE- strep Excellent Camp Negative

Esculin positiveStaph. species ExcellentE. Coli ExcellentKlebsiella pneumoniae ExcellentKlebsiella species ExcellentPseudomonas species ExcellentPseudomonas aeruginosa ExcellentPasteurella ExcellentProteus ExcellentSerratia ExcellentBacillus ExcellentYeast ExcellentMold ExcellentNocardia ExcellentPrototheca ExcellentArcanobacterium pyogenes ExcellentC. bovis Excellent

Udder Health Systems, Inc. FTP\Agar\Inulin agar.doc877-398-1360 Rev: 9/10/2002

Udder Health SystemsInulin Agar

PurposeThis is an indicator agar used at UdderHealth Systems Laboratory fordifferentiating Streptococcus uberis fromother E-streps in isolates from individualcow samples. The agar is also used as aselective agar in bedding samples for theisolation of Streptococcus uberis.

DescriptionThis light purple opalescent agar is prepared from an inulin agar base. The agar hasbeen formulated to differentiate Streptococcus uberis from other E-streps.Streptococcus uberis will ferment the sugar inulin, causing a background yellowingfor Streptococcus uberis and no background color change for other suspect E-Streporganisms. On isolates from commercial dairy clinical specimens, this agar willdifferentiate Streptococcus uberis from other E-Streps with a sensitivity of 94 %and specificity of 92%. The most frequently false positive inulin organism isolatedfrom field specimens is Streptococcus bovis. UHS Inulin agar will inhibit a widevariety of fastidious microorganisms.

Refer to table on back for expected culture responses.

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Udder Health SystemsInulin Agar

Culture Response

Organism Growth ReactionStrep. agalactiae WeakStaph. aureus InhibitedMycoplasma Not ApplicableStrep. dysgalactiae ExcellentStrep. uberis Excellent YellowE- strep ExcellentStaph. species InhibitedE. Coli InhibitedKlebsiella pneumoniae InhibitedKlebsiella species InhibitedPseudomonas species WeakPseudomonas aeruginosa InhibitedPasteurella InhibitedProteus InhibitedSerratia InhibitedBacillus InhibitedYeast InhibitedMold InhibitedNocardia InhibitedPrototheca InhibitedArcanobacterium pyogenes InhibitedC. bovis Inhibited

Udder Health Systems, Inc. FTP\Agar\MacConkey agar.doc877-398-1360 Rev: 1/23/2007

Udder Health SystemsMacConkey Agar

DescriptionOne of the most common causes of environmental mastitis is coliforms which aregram-negative lactose fermenters. This pink agar is a selective media that uses thecharacteristic of growth and lactose fermentation to identify gram-negativeorganisms. Lactose fermentation is expressed on this agar through the productionof a pink colony. E. coli and Klebsiella spp. are the most common lactosefermenting mastitis organisms. Both organisms produce a pink colony. E. colicolonies are pink and generally smaller compared to Klebsiella spp., which arelarger and produce a pink colony with a mucoidal appearance. Non-lactosefermenting organisms (i.e.Pseudomonas species) will also grow on MacConkeyagar, however, the colonies will be colorless or opaque. The antimicrobialcomponents of this agar inhibit the growth of gram-positive and some gram-negative bacteria, such as Pasteurella. When used as a secondary test, the inabilityof Pasteurella to grow on MacConkey agar is the primary characteristic of thisorganism used in its identification.

Refer to table on back for expected culture responses.

PurposeThis agar is used at Udder HealthSystems Laboratory for the detection,selection, and identification of variousgram negative organisms fromindividual cow and environmentalsamples.

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Udder Health SystemsMacConkey Agar

Culture Response

Organism Growth ReactionStrep. agalactiae InhibitedStaph. aureus InhibitedMycoplasma Not ApplicableStrep. dysgalactiae InhibitedStrep. uberis InhibitedE- strep InhibitedStaph. species InhibitedE. Coli Excellent Pink coloniesKlebsiella pneumoniae Excellent Pink mucoid coloniesKlebsiella species Excellent Pink mucoid coloniesPseudomonas species Excellent Colorless coloniesPseudomonas aeruginosa Excellent Green/colorless coloniesPasteurella No GrowthProteus No GrowthSerratia Excellent Pinkish/red colonies*Bacillus InhibitedYeast No GrowthMold Not ApplicableNocardia Not ApplicablePrototheca Not ApplicableArcanobacterium pyogenes InhibitedC. bovis Not Applicable

*Note: Red color can be due to pigment production, not lactose fermentation.

Udder Health Systems, Inc. FTP\Agar\Coagulase Tube Test.doc877-398-1360 Rev: 9/18/2006

Udder Health SystemsCoagulase Tube Test

PurposeThe presence of coagulase enzyme inStaphylococcus organisms isconsidered a key pathogenic featurefor the diagnosis of Staphylococcusaureus. Testing for the presence ofthis enzyme in isolatedStaphylococcus colonies is a standardmethod for differentiating thepathogenic Staph. aureus strain fromother pathogenic and non pathogenicstaphylococcal strains.

DescriptionAnimal strains of Staph. aureus from cows and dogs will frequently produce alarge partial hemolytic zone on blood agar culture indicating its ability to produceBeta toxin. In Bovine Mastitis microbiology, the presence of this “Beta Zone” isconsidered diagnostic for Staph. aureus. In addition to these Beta toxin producingstrains, cultures from Staph. aureus mastitis cows will also show that not all Staphaureus strains are Beta producing strains. Some Staph. aureus strains only producean alpha hemolysin that causes a small clear hemolytic zone on blood agar andsome are non hemolytic. Coagulase testing is the recommended method ofidentifying these non-Beta producing strains. An isolated colony of coagulaseproducing Staph. aureus may produce the gelling reaction in as little as 4 hours.The coagulase enzyme will cause the partial or complete gelling of blood plasma.In the picture above the top tube shows that the plasma is still liquid, indicating anegative result. The bottom tube shows a positive reaction. The plasma in thebottom tube is coagulated and remains adhered to the bottom of the tube when it istilted.

The Udder Health Systems coagulase plasma is shipped in the liquid or frozen stateand is ready to use. It is typically dispensed in approximately 1 ml aliquots toperform the test. Unlike lyophilized rabbit plasma, UHS coagulase plasma does notneed to be reconstituted and is typically lower in test cost than rabbit plasma. It isgood for several years in the frozen state and remains effective for at least 12weeks at refrigerated temperatures.

Refer to table on back for expected test responses.

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Udder Health SystemsCoagulase Test

Culture Response

Organism Growth ReactionStrep. agalactiae ExcellentStaph. aureus * Excellent CoagulationMycoplasma Not ApplicableStrep. dysgalactiae ExcellentStrep. uberis ExcellentE- strep ExcellentStaph. species * ExcellentE. Coli † Excellent False PositiveKlebsiella pneumoniae ExcellentKlebsiella species ExcellentPseudomonas species ExcellentPseudomonas aeruginosa ExcellentPasteurella ExcellentProteus ExcellentSerratia ExcellentBacillus ExcellentYeast ExcellentMold ExcellentNocardia ExcellentPrototheca Excellent

Arcanobacterium pyogenes ExcellentC. bovis Excellent

All of these organisms will grow in the coagulase broth, this growth will produce a cloudyappearance. This is not to be interpreted as a positive reaction, only the coagulation reaction is a positiveresult. The coagulation can be detected in as little as 4 hrs. Typically the reaction is still readable in 24hrs. Some false negatives will occur when coagulation occurs but the clot will lyse again by 24 hrs.

* All Staph species will grow in the coagulase broth, but only Staph. aureus will generate a positivecoagulation reaction. The coagulation can be detected in as little as 4 hrs. Typically the reaction is stillreadable in 24 hrs. Some false negatives will occur when coagulation occurs but the clot will lyse again by24 hrs.

† Some strains of E. coli. can produce the coagulase reaction. A false positive result can occur if theanalyst mistakenly tests an E. coli colony. It is the responsibility of the analyst to only perform this testonly on Staphylococcus genus isolates.

Udder Health Systems, Inc. FTP\Agar\Mycoplasma agar.doc877-398-1360 Rev: 9/11/2002

Udder Health SystemsMycoplasma Agar

PurposeThis is the standard isolation agar used atUdder Health Systems Laboratory forcultivating mycoplasma organisms fromindividual cow and bulk tank milksamples.

DescriptionThis light to medium amber agar is prepared from a purified agar base with theaddition of PPLO broth. This agar has been formulated with inhibitors todiscourage a variety of non-target organisms. The agar is especially formulated toinhibit slow growing contaminating organisms that show up in 10 day incubation.The agar has been tested against University of California Davis mycoplasma agar,with side by side comparisons of field isolates. The agar demonstrated anequivalent or greater recovery of mycoplasma.

Refer to table on back for expected culture responses.

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Udder Health SystemsMycoplasma Agar

Culture Response

Organism Growth ReactionStrep. agalactiae InhibitedStaph. aureus InhibitedMycoplasma Excellent Fried-Egg coloniesStrep. dysgalactiae InhibitedStrep. uberis InhibitedE- strep InhibitedStaph. species InhibitedE. Coli InhibitedKlebsiella pneumoniae InhibitedKlebsiella species InhibitedPseudomonas species InhibitedPseudomonas aeruginosa InhibitedPasteurella InhibitedProteus InhibitedSerratia InhibitedBacillus InhibitedYeast InhibitedMold InhibitedNocardia InhibitedPrototheca InhibitedArcanobacterium pyogenes InhibitedC. bovis Inhibited

Udder Health Systems, Inc. FTP\Agar\MP2 agar.doc877-398-1360 Rev: 9/9/2004

Udder Health SystemsMP2 Agar

PurposeThis is a selective isolation agarused at Udder Health SystemsLaboratory for cultivating grampositive mastitis organisms fromindividual cow milk samples.

DescriptionThis cherry red, agar is prepared from a modified Columbia agar base with theaddition of 5% washed bovine erythrocytes and full strength esculin. Thisselective agar has been formulated to enhance the growth of gram-positivestaphylococci, streptococci and enterococci bacteria while inhibiting the growth ofall gram- negative Enterobacteriaceae and Pseudomonas organisms. The additionof esculin enhances the capability of differentiating Streptococci. CertainStreptococci will hydrolyze esculin, causing a mild background blackening for E-strep and a mild greening or no background coloration for suspect Streptococcusagalactiae and Streptococcus dysgalactiae organisms. All suspect Streptococcusagalactiae organisms should be moved to our special Camp media forconfirmation.

Refer to table on back for expected culture responses.

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Udder Health SystemsMP2 Agar

Culture ResponseUpdated: 1/6/05

Organism Growth ReactionStrep. agalactiae Excellent Esculin negativeStaph. aureus Excellent Hemolytic (beta)Mycoplasma Not ApplicableStrep. dysgalactiae Excellent Esculin variableStrep. uberis Excellent Esculin positiveE- strep Excellent Esculin positiveStaph. species Excellent Hemolytic variableE. Coli InhibitedKlebsiella pneumoniae InhibitedKlebsiella species InhibitedPseudomonas species InhibitedPseudomonas aeruginosa InhibitedPasteurella FairProteus InhibitedSerratia InhibitedBacillus InhibitedYeast n/aMold n/aNocardia n/aPrototheca InhibitedArcanobacterium pyogenes Excellent 24-48 hourC. bovis n/a

Udder Health Systems, Inc. FTP\Agar\TNT2 agar.doc877-398-1360 Rev: 4/7/2008

Udder Health SystemsTNT2 Biplates

PurposeThis biplate is used at Udder HealthSystems Laboratory for selectionand identification of both gram-positive and gram-negativeorganisms.

Description The bright red agar portion of the biplate is prepared from a modified Columbia

base agar with the addition of 5% defibrinated sheep blood, and esculin. Thisselective agar has been formulated to enhance the growth of gram-positivestaphylococci, streptococci and enterococci bacteria while inhibiting the growthof all gram-negative organisms, such as Enterobacteriaceae and Pseudomonas.The addition of esculin enhances the capability of differentiating Streptococci.Certain Streptococci will hydrolyze esculin, causing a mild backgroundblackening. This blackening differentiates E-streps from Streptococcusagalactiae which cannot split esculin. All suspect Streptococcus agalactiaeorganisms should be moved to our special Camp media for confirmation.

The pink agar portion of the biplate is specialized media for the detection ofgram-negative organisms. Since the antimicrobial agents in this agar inhibit thegrowth of gram-positive bacteria, the media focuses on the organisms’ ability toferment lactose. This fermentation causes the bacterial colony to change to apinkish color. Non-lactose fermenting colonies will remain colorless andopaque.

Refer to table on back for expected culture responses.

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Udder Health SystemsTNT2 Biplate

Culture ResponseUpdated: 2/27/08

Gram Positive Agar MacConkey Agar Organism Growth Reaction Growth Reaction

Strep. agalactiae Growth Esculin Negative Inhibited

Staph. aureus Growth Hemolytic Variable Inhibited

Mycoplasma Not Applicable Not Applicable

Strep. dysgalactiae Growth Esculin Variable Inhibited

Strep. uberis Growth Esculin Positive Inhibited

E- strep Growth Esculin Positive Inhibited

Staph. species Growth Hemolytic Variable Inhibited

E. Coli Inhibited Excellent Pink Colonies

Klebsiella pneumoniae Inhibited Excellent Pink Mucoid Colonies

Klebsiella species Inhibited Excellent Pink Mucoid Colonies

Pseudomonas species Inhibited Excellent Colorless Colonies

Pseudomonas aeruginosa Inhibited Excellent Green/Colorless Colonies

Pasteurella Weak No Growth

Proteus Ínhibited No Growth

Serratia Inhibited Excellent Pinkish/Red Pigment

Bacillus Inhibited Inhibited

Yeast Not Applicable No Growth

Mold Not Applicable Not Applicable

Nocardia Not Applicable Not Applicable

Prototheca Not Applicable Not Applicable

A. pyogenes Growth Small Hemolytic Inhibited

C. bovis Not Applicable Not Applicable

Udder Health Systems, Inc. FTP\Agar\Prototheca agar1.doc877-398-1360 Rev. 12/10/2010

Udder Health SystemsPrototheca Agar

PurposeThis specialty selective agar is usedfor the cultivation of Prototheca sp.from individual cow milk, bulktank milk or environmentalsamples.

DescriptionThis whitish translucent agar is prepared from a purified agar base with theaddition of selective agents that inhibit bacterial growth and cultivate Prototheca.This colorless algae forms small dull white opaque colonies on this agar that arereadily observed after 48 hours of incubation. The agar is highly selective and isformulated to inhibit a wide variety of bacterial organisms, and is advantageous insamples likely to contain mixed populations of microorganisms. Side by sidestudies comparing this agar against standard washed cow blood agar withcomposite cow milk samples, bulk tank samples and pooled string samples haveshown substantially increased Prototheca detection. All suspect colonies that growon this media should be confirmed microscopically at 200-400X on a wet mount orwith the use of a stain such as Methylene blue or Gram stain.

Refer to table on back for expected culture responses.

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Udder Health SystemsPrototheca Agar

Culture Response

Organism Growth ReactionStrep. agalactiae InhibitedStaph. aureus InhibitedMycoplasma InhibitedStrep. dysgalactiae InhibitedStrep. uberis InhibitedE- strep InhibitedStaph. species InhibitedE. Coli InhibitedKlebsiella pneumoniae InhibitedKlebsiella species InhibitedPseudomonas species InhibitedPseudomonas aeruginosa InhibitedPasteurella InhibitedProteus InhibitedSerratia InhibitedBacillus InhibitedYeast InhibitedMold WeakNocardia InhibitedPrototheca ExcellentArcanobacterium pyogenes InhibitedC. bovis Inhibited

Udder Health Systems, Inc. Prototheca in Bulk Tank and Cow culture.doc

877-398-1360 NMC 50thAnnual Meeting Proceedings

www.udderhealth.com January 23, 2011

USE OF A SELECTIVE AGAR FOR DETECTION OF

PROTOTHECA IN BULK TANK AND COW MILK CULTURE

A. M. Britten, Jon Cerar, and Murali Gurajala

Udder Health Systems, Inc.

Boise, Idaho, USA

Prototheca species are widely recognized as a very damaging environmental mastitis pathogen.

Dairy managers and veterinarians rely on sensitive tests to detect this organism in Bulk Tank

Culture (BTC), String Cultures (SC) obtained through a QMI sampler (Quality Management Inc.,

Oakdale, MN) and clinical mastitis culture programs. This colorless algae grows readily on

Blood Agar (BA) and this media is commonly used in these culture programs. In BTC and SC,

because the sample always contains a mixed population of organisms, there is concern that the

small Prototheca colonies may be misdiagnosed due to its morphologic similarity to other

mastitis organisms or may be completely overlooked due to competing bacteria. The common

practice in herd culture procedures is to collect an individual composite cow sample (ICC) of the

four quarters from each cow. This also leads to a situation where one is likely to obtain a sample

containing a mixed population of organisms in the culture, due to multiple infected quarters or

contaminating organisms from the skin. The use of Prototheca Isolation Media (PIM)1 has been

advocated as a means of aiding the detection of Prototheca sp. Three investigations are reported

here evaluating the hypothesis that using PIM culture for detection of Prototheca in BTC, SC or

ICC testing procedures would improve detection over conventional BA culture.

Materials and Methods

The two media used for comparison were a commercially prepared washed cow blood agar (BA)

plate and a Prototheca agar (PIM) plate (Udder Health Systems, Inc., Boise, ID). The 3073 bulk

tank samples used in the study were submitted to the laboratory from 432 herds whose tanks

were sampled from 1 to 66 times during a six month period in 2010 as part of routine BTC

services. The 25 SC samples and 887 ICC samples tested in the study came from a single dairy as

part of a Prototheca mastitis problem investigation.

For the BTC and SC testing, an inoculum of .01 mls. of milk sample was collected by a pipetter

and distributed onto a whole plate of BA, and 0.1 ml onto quarter plate of a PIM agar. The larger

inoculation volume on the PIM plate was used to increase sensitivity. The highly selective nature

of this media allows for this as it inhibits nearly all competitive growth in the inoculum. The

plates were incubated and read at 40-48 hours. For the ICC samples, a 3 mm platinum loop was

used to inoculate and streak the specimen onto quarter sections of the BA and PIM plates. These

plates were incubated and read in 48 hours. The ICC milk samples were incubated for 4-6 hours.

For those specimens that had no growth at 48 hours, the plate was re-streaked with the incubated

milk and read again at 96 hrs. All incubation was done at 37oC and 80-85% RH. From each agar,

presumptive Prototheca sp. colonies were selected by colony morphology and confirmed by

microscopic examination according to standard methods2. The presence of at least a single

confirmed colony resulted in that sample being reported as Prototheca positive.

Udder Health Systems, Inc. Prototheca in Bulk Tank and Cow culture.doc

877-398-1360 NMC 50thAnnual Meeting Proceedings

www.udderhealth.com January 23, 2011

Results and Conclusions

Of the 3073 BT samples tested, 314 tested positive for Prototheca on the PIM plate, versus only

49 tanks testing positive on the BA plate (Table 1). On the BT samples the PIM plate detected all

but two of the samples testing positive on the BA plate, but the PIM plate found Prototheca in an

additional 267 tanks not detected on the BA plate. A similar increase in detection rate was seen

when this data was analyzed by herd. Of the 432 herds represented in the tank samples, 89 (21%)

were detected as Prototheca positive on at least one test by either agar. Of these, all but one of

these herds (20%) tested positive on the PIM plate, versus only 27 (6%) on the BA plate. There

were 25 SC samples obtained by QMI on the problem dairy on two different sample days of

which 9 showed Prototheca. All 9 were test positive by PIM plates versus only 3 by BA plates

(Table 2). Of the 887 ICC samples tested, 35 tested positive for Prototheca on the PIM plates,

versus only 22 on BA plates (Table 3). Of the Prototheca positive ICC samples, the PIM plate

detected all but one of the samples that tested positive on the BA plate. In conclusion it was

found that the highly selective PIM will substantially improve Prototheca detection over use of

BA alone based on these investigations on cow milk culture.

Table 1. Test results for detection of Prototheca comparing Prototheca Isolation Media (PIM)

versus Blood Agar (BA) from 3073 bulk tank milk samples.

PIM+ PIM- Totals

BA+ 47 2 49

BA- 267 2757 3024

Totals 314 2759 3073

Table 2. Test results for detection of Prototheca comparing Prototheca Isolation Media (PIM)

versus Blood Agar (BA) from 25 string cultures.

PIM+ PIM- Totals

BA+ 3 0 3

BA- 6 16 22

Totals 9 16 25

Table 3. Test results for detection of Prototheca comparing Prototheca Isolation Media (PIM)

versus Blood Agar (BA) from 887 individual composite cow milk samples.

PIM+ PIM- Totals

BA+ 21 1 22

BA- 14 851 865

Totals 35 852 887

References

1. Pore, R.S. 1973. Selective Media for Isolation of Prototheca. Applied Microbiology Oct.

pp 648-649.

2. National Mastitis Council. Laboratory and Field Handbook on Bovine Mastitis.

Atkinson, WI: W.D. Hoard and Sons Co.

Udder Health Systems, Inc. FTP\Agar\TTBULKTK.doc

A BULK TANK CULTURING PROGRAM FORMONITORING MILK QUALITY AND UDDER HEALTH

A. M. Britten1, and T. Emerson2

1Udder Health Systems, Inc.Bellingham, Washington

2Darigold FarmsSeattle, Washington

A bulk tank culturing service has been available to producers of Darigold Farms in Washington,Oregon, Idaho, and northern California since 19862. The goal of the culture is to identify thespecific types of bacteria that make up the total raw bacteria count. It is a voluntary program andhas grown in popularity over the years. At this point in time over 600 of the 1100 producers inthe cooperative use this service each year.

Sample Procedure

The culture test is performed on a well mixed sample of bulk tank milk from the herd. Typicallythis sample is taken by the milk hauler and placed is a specially marked 4 oz. sample cup at thetime of milk pick up. On some dairies the sample is pulled on a sporadic basis at the request ofthe producer. A large portion of the producers have arranged with the field personnel and thehauler for samples to be automatically taken each month. The samples are frozen and transportedto the testing laboratory usually within 48 hrs.

Testing method

In the laboratory the sample is thawed and plated on four different solid media. Inoculums of .01mls. of milk are plated onto a whole plate of washed cow blood agar, and a portion ofmycoplasma agar and a selective strep. agar plates. Also inoculums of .1 mls. are plated ontoportions of selective staph. agar plates. The non-selective washed cow blood agar is used forgeneral identification and counting of a wide variety of bacteria groups. Bacteria are identifiedaccording to standard methods3.

The selective staph agar is similar in composition to the washed cow blood agar but has 7.5%sodium chloride added. Hemolytic staph colonies from this agar that are positive to the coagulasetest are reported as Staph aureus. The selective strep agar is a modified Edwards4 media withadded esculin, and Staph aureus beta hemolysin. Catalase negative, esculin negative colonieswith CAMP like hemolytic zones around them are reported as Strep. ag. The selectivity of thesemedia and the larger inoculum size allow us to attain greatly improved detection efficiency overthe whole blood agar alone1. The mycoplasma agar is used as a screening test on all bulk tanksfor organisms that will grow on this media. A separate fluorescent antibody test is recommendedfor confirmation of all mycoplasma like colonies grown on this media.

Results

www.udderhealth.com877-398-1360 National Mastitis Council Annual Meeting Proceedings XXXV (1996)

A report is produced and mailed to the producer and the field personnel which shows theidentification and total count of the various bacteria found in his bulk tank sample. With the aidof the fieldman and veterinary consultants, the producer can use the report as an aid in analyzingvarious milk quality and udder health problems in the herd. High somatic cell count tank milk bydefinition comes from herds with significant mastitis problems. The bulk tank culture canfrequently reveal the likely pathogenic causes for these high cell counts. High bacteria counts inbulk tank milk may result from mastitic milk or from contamination, cleaning or coolingproblems. On many occasions identifying the organism causing the high count may help guidethe producer to more specific and appropriate correction efforts. When a major mastitis pathogensuch as Strep ag. or Staph. aureus is causing a high bacteria count, we know disease controlmeasures and abnormal milk diversion is the way to solve the problem. Conversely if organismsin the pseudomonas or bacillus group are too numerous then mastitis is probably not the culpritand attention should be turned toward environmental sanitation.

A special effort has been made to optimize the detection for the major mastitis pathogensStreptococcus agalactiae, Staphylococcus aureus, and Mycoplasma sp. Staph. aureus infection inherds is common and some producers just hope to keep the bulk tank counts for this pathogenlow. Many progressive producers strive to keep the bulk tank counts for all three of these mastitispathogens at zero. The table below shows pathogen detection statistics for a twelve month periodending in 1995.

Table 1. Pathogen Detection from Bulk Tank Culture ProgramHerds Positive % Herds Positive

Strep ag. 31 5Staph aureus 479 81

Mycoplasma sp. 34 6Total herds tested: 589

References

1. Britten, A.M., 1996. Use of a selective agar for improving streptococcus agalactiae detection.Proc. 35 Annual Meeting of the National Mastitis Council.

2. Emerson, T., 1989. Bulk milk bacterial culturing - an aid to quality milk production. Proc. 28Annual Meeting of the National Mastitis Council, pp 49-53.

3. National Mastitis Council. Laboratory and Field Handbook on Bovine Mastitis. Atkinson, WI:W.D. Hoard and Sons Co.

4. Schalm, O.W., Carrol, E.J., Jain, N.C., 1971. Bovine Mastitis. Philadelphia, PA: Lea &Febiger. pp 171-173.

Udder Health Systems, Inc. FTP\Agar\TTSTREP.doc877-398-1360 National Mastitis Council Annual Meeting Proceedings XXXV (1996)

USE OF A SELECTIVE AGAR FOR IMPROVINGSTREPTOCOCCUS AGALACTIAE DETECTION

A. M. BrittenUdder Health Systems, Inc.Bellingham, Washington

Streptococcus agalactiae is widely recognized as a very dangerous mastitis pathogen. Dairymanagers rely on sensitive tests to detect this organism in prevention (bulk tank monitoring) andintervention (test and treat) programs. In bulk tank culture, because the sample is always a mixedculture, there is concern that the small Strep. ag. colonies may be missed because of competingbacteria. The common practice in herd culture procedures is to collect a single composite sampleof the four quarters from each cow. This also leads to a situation where one is likely to obtain amixed culture. Add to this the possibility of organisms from the skin contaminating the cultureand again we have the concern that the Strep. ag. colonies may be missed. The use of selectivestreptococcus agar such as Edward’s2 media has been advocated as a means of aiding thediagnosis of Strep. ag. Two investigations are reported here evaluating the hypothesis that usinga selective strep agar for detection of Strep ag. on bulk tank samples and on individual cowcomposite cultures would, improve detection over conventional blood agar.

Materials and Methods

The two media used for comparison are a washed cow blood agar plate with added esculin (BA)and a selective strep agar plate (E). The E agar is a modified Edward’s media with added esculin,and Staph aureus beta hemolysin. The 5074 bulk tank samples used in the study were submittedto the laboratory from 589 herds whose tanks were sampled from 1 to 57 times during a one yearperiod. The 2120 individual cow composite samples tested in the study came from three herdswith known Strep ag. histories.

For the bulk tank study, an inoculum of .01 mls. from a pipetter was placed onto a whole plate ofBA, and onto half an E agar plate. The plates were incubated and read at 40-48 hours. For theindividual cow composite samples a 3 mm platinum loop was used to streak the specimen onto aquarter section of the BA and E plates. These plates were incubated and read in 24 hours. Themilk samples were incubated for 4-6 hours. For those specimens that had no growth at 24 hours,the plate was re-streaked with the incubated milk.

Streptococcus agalactiae identification was made according to standard methods1. From the BAplate non-esculin strep. candidates were selected for testing for CAMP reaction on a separateCAMP plate. From the E plate, catalase negative, esculin negative colonies with large hemolyticzones around them were selected for re-testing for CAMP reaction on a separate plate. Thepresence of at least a single CAMP positive colony resulted in that sample being reported asStrep. ag. positive.Results

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Of the 5074 bulk tank samples tested, 94 tanks tested positive for Strep. ag.when the E plate wasused to detect the pathogen, versus only 43 tanks testing positive on the BA plate. (Table 1.). Asimilar increase in detection rate was seen when this data was analyzed by herd. Of the 589 herdstested, 31 herds tested positive for Strep. ag. on the E plates, versus only 15 on the BA plates(Table 2.). For the tank samples tested, the E plate always detected the Strep. ag. samples thatwere detected by the BA plate. An even more dramatic difference is seen on the composite cowsamples tested (Table 3.). Of the 2120 composite samples tested, 220 tested positive for Strep.ag. on the E plates, versus only 49 on BA plates. On these samples the E plate detected all butthree of the samples testing positive on the BA plate. Although the degree of difference forcomposite samples was strongly influenced by one herd, the difference was significant in allthree herds. Although the incubation procedure increased the positive detection rates for both theE and BA plates it was only significant for the E plate.

Table 1. Test results for detection of Streptococcus agalactiae comparing a strep selective agar Eversus washed cow blood agar BA from 5074 bulk tank samples.

E+ E- TotalsBA+ 43 0 43BA- 51 4980 5031Totals 94 4980

Table 2. Test results for detection of Streptococcus agalactiae comparing a strep selective agar Eversus washed cow blood agar BA from samples shown in Table. 1. when analyzed by herd.

E+ E- TotalsBA+ 15 0 15BA- 16 558 574Totals 31 558

Table 3. Test results for detection of Streptococcus agalactiae comparing a strep selective agar Eversus washed cow blood agar BA from 2120 cow composite samples from three problem herds.

E+ E- TotalsBA+ 46 3 49BA- 174 1897 2071Totals 220 1900

References

1. National Mastitis Council. Laboratory and Field Handbook on Bovine Mastitis. Atkinson, WI:W.D. Hoard and Sons Co.

2. Schalm, O.W., Carrol, E.J., Jain, N.C., 1971. Bovine Mastitis. Philadelphia, PA: Lea &Febiger. pp 171-173.

Udder Health Systems, Inc. FTP\Agar\Inulin agar tech report.doc877-398-1360 2007

Udder Health Systems Technical ReportInulin Agar Evaluation Study

This study was conducted to test the Udder Health Systems Inulin Agar for its suitability inidentification of Streptococcus uberis organisms from mastitis isolates. Inulin agar is a specialformulation that contains two principal systems that are required to make this media useful foridentification of Strep uberis and differentiating this species from other e-strep mastitis isolatesin cow milk samples. There is an enrichment nutrient system in the agar that allows for thegrowth requirements of these streptococci. The agar uses the sugar inulin as the single source ofcarbohydrate. When organisms that ferment inulin are grown on the agar, there is an indicatorsystem which shows a distinct yellowing of the agar to show that the organism has metabolizedthis sugar. Strep uberis is one of the streptococci that will ferment inulin. It is suggested byUdder Health Systems that this agar be used in a multi-step secondary screening procedure toidentify this pathogen from cow milk samples. The test for inulin fermentation capabilityshould only be done on esculin positive isolates from cow milk in the proposed scheme. In thisscheme if an esculin positive cow milk isolate is Inulin Agar test positive (ferments inulin) thenit is Strep uberis. The goal of the investigation was to estimate the sensitivity or specificity ofthis detection scheme. Sensitivity (the ability of the test to detect Strep uberis) was simplydetermined by comparing the number of isolates that were test positive to the total number ofStrep uberis tested. The Specificity (the ability of the test to correctly identify non-strep uberise-streps) was demonstrated by comparing the number of non-strep uberis organisms that weretest negative to the total number of non-strep uberis tested.

The table below summarizes the data from the study in the form of a truth table.

API RESULTSStrep. uberis Non- Strep uberis

INULIN +93 8 101

INULIN -6 95 101

TOTAL99 103 202

Sensitivity Specificity

94% 92%

A total of 202 isolates of e-streps isolated from mastitic cow milk were included in the study.All the e- streps were speciated using the API rapid strep identification system. Of the 99 Strepuberis isolated tested, 93 of them were inulin test positive on this agar, resulting in a 94%Sensitivity. Of the 103 non-strep uberis organisms, 95 of them were test negative resulting in a92% Specificity. In this study, the 8 organisms that were False Positive (inulin positive non-strep uberis) included 6 Strep bovis, 1 Ent faecium, 1 Aerococcus sp. The 6 False Negativeorganisms were by definition inulin negative Strep uberis.

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877-398-1360 Rev: 1/11/2005

Udder Health Systems Technical Report

2004 New Mycoplasma Agar Comparison Study

This study was conducted to assess the suitability of Udder Health System’s new ultra clearMycoplasma agar in isolating mycoplasma organisms from bovine milk. The two agars in thestudy were the new ultra clear Mycoplasma agar plates manufactured by Udder Health Systems,Inc. Bellingham, Washington and the comparison mastitis mycoplasma agar plates manufacturedby the University of California at Davis.

Mycoplasma isolation agar is a special formulation that contains two principal systems that arerequired to make this media useful for detection of mastitis causing mycoplasma from milksamples. There is an enrichment nutrient system, typically including serum, which allows for thespecial growth requirements of these fastidious, slow growing organisms. There are also inhibitorsystems, which include antibiotics, to discourage non-mycoplasma growth. Typically non-targetorganisms may be present in individual cow milk samples and will always be present in bulk tanksamples. To be successful in detecting mycoplasma organisms, these non-target species must beinhibited. If they do grow on the mycoplasma agar, they may overwhelm and obscure themycoplasma growth and prevent their detection.

The goal of this study was to determine if there were any differences in the sensitivity orspecificity of the two agars. Sensitivity (the ability to detect the mycoplasma in the specimen) wassimply determined by comparing the organism counts based on plating the same specimen on awhole plate of each agar. The Specificity of the agar (the ability to only grow mycoplasmacolonies and not grow competing bacteria) was demonstrated by the number of plates showingcontaminating growth on each agar.

The following pages contain the raw data from the study. The samples used had been previouslyidentified as containing mycoplasma, and had been stored frozen for up to one year before thestudy was initiated. Samples were identified by Sample number and Source (cow/tank). Eachsample was plated in duplicate on UHS Media agar and UC Davis Media agar. The total numberof mycoplasma colonies growing on each plate is shown in the column totals for each agar. It isclear from the raw data that the UHS Media is as sensitive or more sensitive than the UC Davismedia is.

We also looked at natural contaminants that grew on the two agars from these field specimens.These are shown in the columns labeled Cont. In these data an advantage is shown in the inhibitorsystem in the UHS media, which we believe is particularly important for laboratories using a 10-day incubation (which UHS recommends). Contaminants showed in five of the UC Davis platesbut only three of the UHS plates. Contaminant inhibition is important in selective agars likemycoplasma agar. Non-mycoplasma growth may interfere with the detection of low numbers ofmycoplasma that may show up between day 5 and 10 of incubation.

Udder Health Systems, Inc. FTP\Agar\Myco agar 2004 tech report.doc

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Udder Health Systems versus UC Davis MediaNew UHS Media UC Davis Media New UHS Media UC Davis Media

Sample Source Plate 1 Plate 2 Cont. Plate 1 Plate 2 Cont. Sample Source Plate 1 Plate 2 Cont. Plate 1 Plate 2 Cont.1 Cow 0 0 1 1 51 Cow 1 3 2 02 Cow 999 999 999 999 52 Cow 0 0 0 03 Cow 0 0 0 0 53 Cow 0 0 0 04 Cow 0 0 0 0 54 Cow 0 0 0 05 Cow 1 4 1 3 55 Cow 151 102 45 386 Cow 0 0 0 0 56 Cow 0 0 0 07 Cow 476 437 268 295 57 Cow 999 999 999 9998 Cow 999 999 999 999 58 Tank 0 19 8 09 Cow 97 113 60 105 59 Cow 999 999 999 99910 Cow 3 0 0 0 60 Tank 0 0 0 011 Cow 30 52 38 25 61 Cow 0 0 0 012 Cow 999 999 999 999 62 Cow 0 0 0 013 Cow 12 54 4 4 63 Cow 0 0 0 014 Cow 999 999 999 999 64 Cow 0 0 15 10315 Cow 150 187 0 20 65 Cow 999 999 999 99916 Cow 12 7 12 16 66 Cow 999 999 254 40317 Cow 0 8 0 0 67 Cow 999 999 999 99918 Cow 38 7 176 19 68 Cow 26 12 17 3319 Cow 75 65 55 50 69 tank 8 8 12 1220 Cow 999 999 999 999 70 tank 0 0 1 121 Tank 0 0 0 0 71 tank 0 0 0 022 Tank 0 0 0 0 72 tank 25 25 20 2023 Cow 999 999 999 999 73 tank 10 10 2 224 Cow 0 0 0 0 74 tank 0 0 0 025 Cow 0 0 0 0 75 tank 999 999 200 20026 Cow 999 999 999 999 76 tank 8 8 0 027 Cow 74 51 82 81 77 tank 40 40 15 1528 Cow 999 999 999 999 78 tank 4 4 2 129 Cow 32 36 19 18 79 tank 0 0 3 0 0 330 Cow 351 372 324 415 80 tank 30 30 20 2031 Cow 0 0 0 0 81 tank 30 30 12 1232 Cow 0 0 0 0 82 tank 5 10 2 133 Tank 0 0 0 0 83 tank 0 0 0 0 534 Cow 999 999 999 999 84 tank 10 10 20 2035 Cow 999 999 999 999 85 tank 50 50 0 036 Tank 0 0 0 0 86 tank 0 0 15 20 1537 Tank 0 0 0 0 87 tank 25 25 4 438 Tank 0 0 0 0 88 tank 0 0 999 0 0 99939 Cow 0 0 0 0 89 tank 50 50 50 5040 Cow 999 999 999 999 90 tank 0 0 100 0 0 10041 Cow 0 0 0 0 91 tank 0 0 0 042 Cow 50 61 44 42 92 tank 0 0 0 043 Cow 999 999 999 999 93 cow 0 0 0 044 Cow 0 0 0 0 94 cow 3 3 4 245 Tank 0 0 0 0 95 cow 0 0 0 046 Cow 0 0 0 0 96 cow 0 0 0 047 Cow 0 0 0 0 97 cow 0 0 0 048 Cow 999 999 0 0 Column Totals 20858 20874 3 17788 18035 549 Cow 0 0 0 0 Sum of double plate: Plate 1 Plate 2 Cont. Plate 1 Plate 2 Cont.50 Cow 0 0 0 0 New UHS Media UC Davis Media

FTP\Agar\Myco study data 2004.docUdder Health Systems, Inc.877-398-1360www.udderhealth.com