Human , cattle and goat blood as substitutes for sheep blood in blood-supplemented culture media

Introduction and Objective: Sheep blood is the recommended type of blood for supplementation of agar media. In Sri Lanka, due to lack of availability of sheep blood, expired citrated human blood is used which gives poor haemolysis and causes difficulties in identification of some organisms. In addition, human blood contains antibodies and other antibacterial factors including antibiotics which may inhibit bacterial growth. Human blood may also contain blood borne pathogens which could be a risk for laboratory staff. The objective of this study was to explore available alternatives in the Sri Lankan setting. Methods: Clinical isolates and standard strains of Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus pneumoniae and Haemophilus influenzae were tested for growth, identification characteristics and antibiotic susceptibility on human, cattle, goat and sheep blood agars. The performances were compared. Identification of Listeria monocytogenes as well as Streptococcus agalactiae was carried out using the CAMP test. Results: All tested organisms gave similar isolation rates at the tested dilutions in the four tested agar plates. Human blood gave noticeably smaller colonies. S. pyogenes and S. agalactiae gave equally large zones of beta-haemolysis and S. pneumoniae gave obvious alpha-haemolysis on all animal blood agar plates. Both types of haemolysis were faint on human blood. Typical arrow head shape haemolysis for S. agalactiae and match-head shape haemolysis for Listeria were seen in the CAMP test on the three animal blood agar plates whereas human blood gave negative results. All blood agar plates gave comparable positive results in the satellitism test for H. influenzae. There was no difference in bacitracin and optochin sensitivity tests for identification of S. pyogenes and S. pneumoniae respectively. Inhibitory zones were unreadable when antibiotic susceptibility was done for H. influenzae on goat and cattle chocolate agar. Sheep and human chocolate agar were inferior to Haemophilus test medium. ABST results were equivalent but goat blood gave hazy, irregular margins for other organisms.


Introduction
Artificial culture media play a very important role in microbiological laboratories.Robert Koch (1843-1910) is considered as the father of artificial culture media. 1 Agar media supplemented with 5% blood are widely used in laboratories for routine isolation, identification and antibiotic susceptibility testing (ABST) of bacterial pathogens. 1,2Some fastidious bacteria such as Neisseria and Haemophilus species require "X" factor (Haemin) and "V" factor (Nicotinamide adenine dinucleotide -NAD) which are found in blood cells.Hemin (factor X) is available from non-haemolysed as well as haemolysed blood cells.The lysis of RBC during the heating process releases intracellular coenzyme NAD (V Factor) into the agar.The heating process also inactivates the growth inhibitors in blood. 3ood supplementation allows the visualization of haemolysis which assists identification of some organisms.Streptococcus species give different types of haemolysis (β-haemolysis and αhaemolysis). 4Blood supplementation minimizes misidentification of colonies, thereby reducing specimen processing cost and turnaround time, and helps accuracy of reports. 4Defibrinated sheep blood is largely preferred because it prevents growth of some nonpathogenic commensals (eg: Haemophilus haemolyticus) due to growth inhibitors and also gives the best haemolytic patterns. 1Horse blood has also been recommended and is used in some countries. 4However, in many developing countries, including Sri Lanka, sheep and horse blood are difficult to obtain due to unavailability of these animal sources. 5,6Buying these blood products from commercial sources is not feasible due to the high cost.Citrated human blood is therefore used in many resource poor settings, despite its many shortcomings. 2,6Human blood is not generally recommended for enrichment of agar media because of poor bacterial isolation rates and inability to demonstrate proper organism characteristics. 1,2,6Presence of antibodies, antibiotics and various inhibitory components in human blood can account for this. 2 The haemoglobin level of animals such as sheep and cattle are around 8-16 mg/dl 7 in comparison with the haemoglobin level of human red cell concentrate (20 mg/dl).Human blood agar therefore gives a darker colour compared to other animal blood agars, which makes observation of colony characteristics a difficult task.
The CAMP test is commonly used for identification of Streptococcus agalactiae and it is useful identification test for Listeria monocytogenes.The CAMP reaction depends on the synergistic haemolytic activity of two factors; CAMP factor and sphingomyelinase.The CAMP factor is secreted by group B streptococci (GBS) and is a protein with exotoxin and pore-forming properties which are suggested to be important for GBS pathogenesis.Sphingomyelinase is secreted by Staphylococcus aureus strains. 2,8As the sphingomyelin content of human and rabbit blood cells is low, they do not support the CAMP reaction well.The sphingomyelin content of sheep, cattle and goat blood is high, and these blood types support the CAMP reaction well. 2,9e use of human blood is associated with safety risk to laboratory personnel, especially due to transmission of blood-borne viral infections such as hepatitis B, C and HIV and is therefore considered unsuitable for use in clinical diagnostic laboratories.Due to these drawbacks of human blood supplemented media, a suitable alternative is needed in countries where use of sheep blood is not feasible.In Sri Lanka expired citrated human blood obtained from blood banks is commonly used for preparation of media.Taking into consideration the importance of proper blood enriched media in laboratory practice, it is a timely need to look for a suitable substitute.This study was carried out to look for a better source of blood which is affordable and freely available to be used for bacteriology in Sri Lanka.
The objective of the study was to compare the performance of citrated human, cattle, goat and sheep blood as enrichment substance in blood supplemented culture media.These two animal species are commonly available in Sri Lanka, making them a credible option to be considered for this purpose.

Methods
This is a laboratory based descriptive cross-sectional study, carried out in the Department of Microbiology, Faculty of Medical Sciences, University of Sri Jayewardenepura.All procedures were performed according the laboratory manual of the Sri Lanka College of Microbiologists, 2011. 10 Citrated sheep, cattle and goat blood were collected from healthy animals (following examination by a veterinary surgeon) who had no recorded history of blood borne disease and had not received an antibiotic within the previous three months.Blood collection was performed under strict aseptic conditions by experienced professionals and transported in a cold box.Citrated human blood was obtained from the National Blood Transfusion Service.5% blood and chocolate agar, blood Mueller-Hinton agar and Haemophilus Test Medium (HTM) were prepared following standard microbiological methods. 10 of prepared culture plates were incubated overnight at 35 °C for sterility.Standard stock cultures of Streptococcus pyogenes (ATCC 12384), Streptococcus agalactiae (ATCC 12386), Streptococcus pneumoniae (ATCC 49619), Haemophilus influenzae (ATCC 49247) and two clinical isolates each of these four organisms (confirmed identification using standard biochemical tests and/or API kits) were tested and compared for growth, identification characteristics and antibiotic susceptibility on the four different types of blood agar.Three clinical isolates of Listeria monocytogenes (confirmed identification using standard biochemical tests and motility test) were used for the CAMP test.
Colony count, colony size and morphology on each type of blood agar were checked.

Colony count
Each of the above bacterial strains from an overnight culture was inoculated in Brain Heart Infusion Broth (BHI) to make a suspension (turbidity~0.5McFarland standard) and serially diluted in BHI.The neat suspension (~0.5 McFarland standard) and1/10 and 1/100 dilutions of the neat (approximately 10 8 , 10 7 and 10 6 CFU/ml) were used for testing.Agar plates were inoculated using a calibrated 1 µl bacteriological loop and incubated overnight at 35 °C in 5-10% CO2.All tests were done in duplicate.The colony count was given as the average of the two readings in units of CFU/ml.

Colony morphology
Sizes of two separated colonies were measured in millimeters and the average taken.Colony morphology was checked by the naked eye.Haemolytic patterns (α, β) for Streptococcus sp. with diameters of the haemolytic zones (mm) were recorded.

Antibiotic susceptibility testing (ABST)
ABST was done according to the guidelines by Clinical Laboratory Standard Institute (CLSI), 2012. 11For Haemophilus influenzae, ABST was performed on Haemophilus Test Medium (HTM) in addition to the four test chocolate agar plates and zones of inhibitions compared.

Morphological characteristics
The colony sizes of all clinical isolates of Streptococcus pyogenes were the same on all blood types.The ATCC strain (12384) however gave very small colonies (<1 mm) on human blood agar (HBA) with minimal haemolysis.
The ATCC strain (12386) of Streptococcus agalactiae gave <1 mm colonies in all four blood agar plates while the clinical isolates grew poorly (<1 mm) only on HBA.Clinical isolates and the ATCC strain gave good β haemolysis on the 3 animal blood agar plates.Although the ATCC strain also gave good β haemolysis on HBA, the clinical isolates performed poorly (<1 mm).
Colony sizes of both clinical isolates and ATCC strains (49619) of Streptococcus pneumoniae were larger (1 mm) on cattle blood agar (CBA) than on the other 3 blood agar plates (<1 mm).
The ATCC strain (49619) gave larger α haemolytic zones (2-3 mm) on the animal blood agar plates in comparison with the clinical isolates (1-2 mm).α haemolysis was negligible (<1 mm) on HBA for all isolates.Beta haemolysis and alpha haemolysis were obvious on SBA, GBA and CBA but faint on HBA as shown in Figures 1 and 2.

Haemophilus influenzae
Clinical isolates and ATCC strains gave 1 mm colonies in Sheep Chocolate Agar (SCHA) and Human Chocolate Agar (HCHA) whereas on Cattle Chocolate Agar (CCHA) and Goat Chocolate Agar (GCHA) they were powdery colonies (Figure 3).

Identification tests
All clinical and standard strains of S. pyogenes were sensitive to 0.04U bacitracin and S. pneumoniae were sensitive to 5 µg optochin in all four blood types.S. agalactiae gave obvious arrow head shaped haemolysis on SBA, CBA, and GBA in CAMP test but this was absent in HBA.Listeria gave match head shaped haemolysis on all types of animal agar and was absent in HBA (Figure 4).For Haemophilus influenzae, all four blood types gave satellitism.However, performance of GBA and HBA were poor.

Antibiotic Sensitivity Testing (ABST)
Inhibition zone sizes of standard strains of Streptococcus sp. were within the recommended range and clinical isolates of all these organisms gave similar results on all 4 types of blood Mueller-Hinton agar (BMHA) for all tested antibiotics (Figure 5).As the H. influenzae strains did not grow properly on CCHA and GCHA, the test was repeated, with similar results.
The inhibition zone sizes of the standard strain of H. influenzae were within the recommended range on HTM for all antibiotics.However, they were not within the recommended range for cefotaxime on SCHA and for ciprofloxacin and cefotaxime on HCHA.
There were no zones of inhibition (ZOI) of clinical isolates I and II with co-amoxiclav on HCHA.However, these isolates were sensitive on HTM and SCHA.Although the other ABST results were same, zone diameters on HCHA and SCHA were smaller than zone sizes of HTM.
Hazy margins of the zones of inhibition were obtained with GBMHA (figure 5) which made reading of the zone diameter difficult.Optochin and bacitracin sensitivity tests as well as ABST results for group A and B streptococci and pneumococci were similar in all four types of blood Mueller-Hinton agar (BMHA).All standard isolates gave zone diameters within the recommended range and clinical isolates were similar in their zone size interpretation results.
The drawback of GCHA and CCHA is that they cannot be used for the ABST of H. influenzae due to poor growth, resulting in unreadable inhibitory zones.HTM gave the best results for ABST of Haemophilus influenzae in this study.In addition, accurate reading of the ZOI was difficult with goat blood supplemented MHA (Figures 5 and 6).
When comparing goat and cattle blood agar, cattle blood agar is better than goat blood agar for the CAMP test which is useful in identification of Group B streptococcus and Listeria because GBA gave arrow head shaped synergistic haemolysis with hazy margins in CAMP test.CBA gave typical arrow head shape with clear-cut margins.

Limitations of the study
Pig blood is another possible source which was considered for this study.However, phlebotomy of pigs was difficult due to their thick fat layer.Only about 10ml of blood could be obtained from one pig at a time which would make it difficult to obtain sufficient supplies for routine laboratory services.High volumes of blood could be obtained from slaughter houses.However, with current methods of slaughter, ensuring a sterile blood supply would not be possible.This source was therefore removed from the study.This is only a preliminary study which was done using a small number of organisms.To confirm the results, further studies are needed with larger numbers of different organisms.Further, the different types of blood agar require testing for isolation of organisms from different clinical specimens as isolation might vary according to the site of the specimen.
CAMP (Christie-Atkins-Munch-Peterson) test for Streptococcus agalactiae and Listeria monocytogenes and satellitism test for Haemophilus influenzae were performed. 100.04U bacitracin and 5µg optochin sensitivity tests were done for Streptococcus pyogenes and Streptococcus pneumoniae respectively.

Figure 4 :
Figure 4: CAMP test on different types of blood agar

Table 1 :
Colony count (CFU/ml) of tested organisms on all four blood agars at different dilutions

Table 2 :
Colony morphology of organisms on different types of agar