Hair sheep blood, citrated or defibrinated, fulfills all requirements of blood agar for diagnostic microbiology laboratory tests

Abstract

Background: Blood agar is used for the identification and antibiotic susceptibility testing of many bacterial pathogens. In the developing world, microbiologists use human blood agar because of the high cost and inhospitable conditions for raising wool sheep or horses to supply blood. Many pathogens either fail to grow entirely or exhibit morphologies and hemolytic patterns on human blood agar that confound colony recognition. Furthermore, human blood can be hazardous to handle due to HIV and hepatitis. This study investigated whether blood from hair sheep, a hardy, low-maintenance variety of sheep adapted for hot climates, was suitable for routine clinical microbiology studies.

Methods and findings: Hair sheep blood obtained by jugular venipuncture was anticoagulated by either manual defibrination or collection in human blood bank bags containing citrate-phosphate-dextrose. Trypticase soy 5% blood agar was made from both forms of hair sheep blood and commercial defibrinated wool sheep blood. Growth characteristics, colony morphologies, and hemolytic patterns of selected human pathogens, including several streptococcal species, were evaluated. Specialized identification tests, including CAMP test, reverse CAMP test, and satellite colony formation with Haemophilus influenzae and Abiotrophia defectiva were also performed. Mueller-Hinton blood agar plates prepared from the three blood types were compared in antibiotic susceptibility tests by disk diffusion and E-test.

Conclusions: The results of all studies showed that blood agar prepared from citrated hair sheep blood is suitable for microbiological tests used in routine identification and susceptibility profiling of human pathogens. The validation of citrated hair sheep blood eliminates the labor-intensive and equipment-requiring process of manual defibrination. Use of hair sheep blood, in lieu of human blood currently used by many developing world laboratories and as an alternative to cost-prohibitive commercial sheep blood, offers the opportunity to dramatically improve the safety and accuracy of laboratory diagnosis of pathogenic bacteria in resource-poor countries.

Figure 1. Comparison of red blood cells from sheep, human, and expired blood bank blood.

(A and B) Sheep red blood cells (A) are smaller in size compared to human red cells (B). (C) Human red blood cells after >45 days of storage in the blood bank demonstrate significant formation of echinocytes, spherical spiculated cells, that are distinctly different from the biconcave disk of normal red cells. All panels are shown with the same scale as indicated in (C).

Figure 2. Identification of bacteria by specialized microbiological tests.

(A) The CAMP test for identification of Group B streptococcus (horizontal streak, GBS) demonstrates an arrowhead shaped area of enhanced hemolysis when grown near S. aureus (vertical streak, SA). (B) The reverse CAMP test for identification of Arcanobacterium haemolyticum demonstrates an indentation in the linear area of hemolysis by S. aureus (vertical streak, SA) when it grows adjacent to A. haemolyticum (horizontal streak, AH). (C) The satellite test for identification of Haemophilus influenzae demonstrates growth of the organisms as small satellite colonies (arrowhead, H. flu) adjacent to S. aureus (vertical streak, SA). Similar satellite colony formation around S. aureus was seen for Abiotrophia defectiva. Similar results were seen on both defibrinated and citrated hair sheep blood agar; results are shown on citrated hair sheep blood agar plates.

Figure 3. Use of hair sheep blood agar in antibiotic susceptibility testing.

(A) Determination of MIC values for penicillin by E-test for a resistant (left) and a susceptible (right) strain of S. pneumoniae. (B) The D-test demonstrates inducible clindamycin resistance in a S. aureus strain carrying an inducible erm gene. A “D”-shaped area of clearing is seen due to increased growth around the clindamycin disk (labeled “C”) where it lies closest to the erythromycin disk (labeled “E”). Similar results were seen on both defibrinated and citrated hair sheep blood agar; results are shown on citrated hair sheep blood agar plates.