64 

The Potential Use of EDTA as an Alternative to Defibrination in 

Preparing Blood Agar Plates with Human AB Blood Type on 

Staphylococcus aureus Culture 
 

Dora Dayu Rahma Turista1,2, Eka Puspitasari2, Fanny Kurnanda2 
 

 
1Biology Education Departement, 

Faculty of Teacher Training and 

Education, Mulawarman 

University, Samarinda, East 

Borneo, Indonesia 

2Departement of Medical 

Laboratory Technology, STIKes 

Hutama Abdi Husada, 

Tulungagung, East Java, Indonesia 

Correspondence: 

Dora Dayu Rahma Turista,  

Jl. Kuaro, Gunung Kelua, 

Samarinda Ulu, Paser, East Borneo, 

Indonesia 

Zip Code: 75119 

 

Email: 

doraturistaofficial@gmail.com 

 

Received: February 6th, 2021 

Revised: March 8th, 2021 

Accepted: March 31th, 2021 

Published: April 28th, 2021 

 

DOI: 10.33086/ijmlst.v3i1.1923 

 

 

 Abstract 

Blood Agar Plates (BAP) are composed of blood as one of 

the compositions. Sheep’s blood is usually used, but since it 

is difficult to be obtained, human AB blood type was used 

as an alternative. In preparing BAP, blood is defibrinated to 

lyse the blood clotting factors. Blood clots can also be 

prevented by adding anticoagulants, such as 

ethylenediaminetetraacetic acid (EDTA). This study aims to 

investigate the potential use of EDTA as a substitute for 

defibrination in preparing BAP with human AB blood type. 

This study employed a completely randomized design with 

true experimental method using Staphylococcus aureus as 

the sample. The parameters were the number of colonies, 

types of hemolysis, and hemolysis zone. The results showed 

that the S. aureus grown on BAP with EDTA-human AB 

blood type was 64 colonies (mean), produced β-hemolytic 

pattern, and 6 mm hemolytic zone. In contrast, the S. aureus 

grown on BAP with defibrinated human AB blood type 

showed 82 colonies (mean), β-hemolytic pattern, and 5 mm 

hemolytic zone. There were significant differences in the 

number of colonies (0.000 < α) and hemolytic zones (0.02 < 

α). However, there was no difference in the hemolysis type 

(both treatments produced β-hemolysis). EDTA was 

possible to be used as a substitute for defibrination in 

preparing BAP to assess the hemolysis type of S. aureus, but 

it might not be able to be used as a benchmark for counting 

the number of colonies and determining the hemolysis zone 

of S. aureus. 

Keywords 

AB Blood Type, Blood Agar Plates, Defibrination, EDTA, 

Staphylococcus aureus 

 
  

 

This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted 

use, distribution, and reproduction in any medium, provided the original work is properly cited. ©2021 by author. 

 

 

mailto:doraturistaofficial@gmail.com
https://journal2.unusa.ac.id/index.php/IJMLST/article/view/1923/version/2407


 

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INTRODUCTION 

Blood Agar Plates (BAP) are universal 

culture media for growing various bacteria 

and also can be used as a differential medium 

to differentiate hemolytic bacteria from non-

hemolytic bacteria. In general, BAP contains 

defibrinated mammalian blood (1). 

Defibrination refers to the process of 

removing clotting factors from blood to allow 

the perfect mixture of blood and medium. 

Blood clots formation can also be prevented 

by adding anticoagulants, such as 

ethylenediaminetetraacetic acid (EDTA) (2). 

EDTA is frequently used since it is unable to 

distort blood cells (3). 

BAP are also used to differentiate 

pathogenic bacteria based on the hemolytic 

patterns (4). Standard BAP are commonly 

prepared using sheep’s blood. However, 

since ensuring the sterility of sheep venous 

blood after the collection process is 

challenging, human A, B, O, or AB blood 

types can be used as alternatives (1). BAP 

with human AB blood type offers easier 

identification of Staphylococcus aureus due 

to its wider hemolytic zone when compared 

to BAP with other blood types (1).  

S. aureus can induce red blood cells 

(RBC) lysis by four hemolysis types (α, β, γ, 

and δ). Hemolysis is observed on the 

appearance of a clear zone around the 

bacterial colony on the BAP. Hemolysis is 

caused by a toxin called hemolysin. It is one 

of the virulence factor of S. aureus. It also 

determines the virulence factor of coagulase 

positive staphylococci (CPS) and coagulase 

negative staphylococci (CNS) (5). It plays a 

role in bacterial invasion and also helps in 

disengaging from immune response (6).  

EDTA has long served as an 

anticoagulant with competitive advantages as 

opposed to other anticoagulants. Its major 

advantage is that it does not promote blood 

cells distortion and hence an excellent option 

for most hematological tests (3). To the best 

of our knowledge, the use of EDTA as a 

substitute for defibrination in preparing BAP 

with human AB blood type as culture media 

for the growth of S. aureus has never been 

conducted. Based on this basis, this study 

aims to investigate the potential use of EDTA 

for such usage. 

 

MATERIALS AND METHODS  

The materials were Blood Agar Base 

(Merck) media, human AB blood type, 

EDTA vacutainer tube, pure culture of S. 

aureus, NaCl 0.9%, and a 0.5 McFarland 

solution. A completely randomized design 

was employed in this study. The S. aureus 

pure culture was obtained from the 

Microbiology Laboratory collection (STIKes 

Hutama Abdi Husada Tulungagung, 

Indonesia). Human AB blood type was 

collected from sterilized venous blood. For 

the control group, the blood was defibrinated 

manually using marbles, while for the 

experimental group, it was directly collected 



 

 

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using an EDTA vacutainer tube (1.8 mg/mL 

blood). After the collection, the blood was 

prepared for making BAP. S. aureus was 

inoculated on NaCl 0.9% and the turbidity 

was matched to a 0.5 McFarland solution for 

density comparison of a bacterial suspension 

with 1.5 x 108 CFU/mL. T streak method was 

used to inoculate S. aureus from NaCl 0.9% 

on BAP. S. aureus was incubated for 48 

hours, then the type of hemolysis was 

observed by measuring the hemolytic zone. 

The S. aureus colonies were also counted. 

The results were analyzed using MANOVA 

with 0.05 significance level. 

  

 

RESULTS 

The colonies of S. aureus were round, 

small, smooth, convex, and white after 48 

hours of incubation on the BAP with EDTA-

human AB blood type. In contrast, when 

grown on BAP with defibrinated human AB 

blood type, S. aureus colonies were medium, 

smooth, convex, and white. The growth of S. 

aureus on the BAP, both with EDTA-human 

AB blood type and defibrinated human AB 

blood type, was observed based on three 

parameters: a) the number of colonies; b) 

zones of hemolysis; and c) types of hemolysis 

(Table 1). Analysis using MANOVA for 

number of colonies and hemolytic zones 

showed that the P-values were 0.000 and 

0.02, respectively.  

 

Table 1. The growth of S. aureus on the BAP with EDTA-human AB blood type and defibrinated 

human AB blood type. 
 

Blood Type 
Number of Colonies Hemolytic Zones (mm) 

Hemolysis Types 

(α, β, γ, or δ) 

1 2 3 1 2 3 1 2 3 

EDTA-human 

AB Blood Type 
62 65 65 6 6 6 β β β 

Defibrinated 

Human AB 

Blood Type 

82 84 80 5 5 5 β β β 

 

DISCUSSION 

Carbohydrates in blood are the main 

sources of nutrition to support bacterial 

growth. Blood can boost the growth of certain 

bacteria (7). Defibrination is only intended to 

remove blood clotting factors in erythrocytes 

rather than to eliminate the nutritional content 

(4). The mean number of S. aureus colonies 

grown on BAP both with EDTA-human AB 

blood type and defibrinated human AB blood 

type is depicted in Figure 1.   

 



 

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Figure 1. The mean number of S. aureus colonies grown on BAP both with EDTA-human AB 

blood type and defibrinated human AB blood type. 

 

The P-value of number of colonies was 

0.000, indicating that there was a significant 

difference between the number of S. aureus 

colonies that grow on BAP with EDTA-

human AB blood type and BAP with 

defibrinated human AB blood type. The 

number of S. aureus colonies grown on BAP 

with defibrinated human AB blood type was 

higher than grown on BAP with EDTA-

human AB blood type. This occurs since 

EDTA has inhibitory power, although it is 

very low. 

In a previous study, the addition of 

EDTA to egg white lysozyme extract reduced 

the number of S. aureus and Salmonella 

typhosa colonies (8). Na-EDTA combined 

with nisin at certain concentrations and pH 

also inhibited the growth of Gram-positive 

and Gram-negative bacteria (9). Combination 

of EDTA/tromethamine and oxytetracycline 

did not significantly affect Staphylococcus 

hominis, whereas EDTA/tromethamine with 

several other antibiotics had low inhibitory 

effects on other antibiotic-resistant bacteria 

(10). EDTA is able to inhibit the growth of S. 

aureus because it can damage the membrane 

of bacterial cells. Bacterial cell membranes 

consist of various types of lipids. The types 

of lipid in each bacteria (even in one species) 

are different. They differ due to 

environmental conditions (11). EDTA is 

believed to have a detergent-like mechanism 

to the chelating mechanism because the 

solution of the tetrasodium salt in EDTA can 

dissolve grease (12,13,14). This alkalinity, 

however, does not occur in low 

concentrations (15). This study underlines 

the abundance of S. aureus colonies which 

were able to grow on BAP containing EDTA 

since there was only a small amount of EDTA 

in the blood (1.8 mg/mL blood). The 

concentration used in this study is extensevily 

used in clinical laboratories for chemical 

blood assay. 

The mean of S. aureus hemolytic zones 

grown on BAP both with EDTA-human AB 

blood type and defibrinated human AB blood 

type is depicted in Figure 2. 

64

82

0

20

40

60

80

100

EDTA AB Defibrinated AB

N
u
m

b
e
r 

C
o
lo

n
ie

s 
in

 

T
h
e
 P

la
te

Blood Type



 

 

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Figure 2. The mean of S. aureus hemolytic zones grown on BAP both with EDTA-human AB 

blood type and defibrinated human AB blood type. 

 

S. aureus colonies grown on BAP with 

EDTA-human AB blood type showed wider 

hemolytic zones compared to those grown on 

BAP with defibrinated human AB blood 

type. The complete hemolytic zones could 

only be observed after 48 hours of incubation. 

A 24-hours incubation did not display any 

hemolytic zones on all plates. Bacterial 

colony characteristics on culture media after 

48 hours of incubation are easier to 

distinguish (16). Hemolytic zones are the 

clear areas around the bacterial colonies that 

grow on BAP. They are determined by lifting 

the agar plates to a light source coming from 

behind (transmitted light), and then the 

diameter is measured by a ruler in millimeters 

(17).  

The P-value of hemolytic zones was 

0.02, indicating that there was a significant 

difference between S. aureus colonies grown 

on BAP with EDTA-human AB blood type 

and those grown on BAP with defibrinated 

human AB blood type. S. aureus colonies on 

BAP with defibrinated human AB blood type 

grew more than those on BAP with EDTA-

human AB blood type. S. aureus was able to 

grow and produce hemolytic zones in both 

BAP with EDTA-human AB blood type and 

BAP with defibrinated human AB blood 

type. Previous study showed that S. aureus 

can grow well in BAP with all of the human 

blood types (A, B, AB, and O) (1). This is 

because the morphology and nutrient content 

in the blood are consistent (18).  

Hemolysis type of S. aureus grown in 

BAP both with EDTA-human AB blood type 

and defibrinated human AB blood type are 

presented in Figure 3. Both treatments 

showed the same type of hemolysis which 

was β-hemolysis. In a previous study, S. 

aureus was able to produce β-hemolysis 

when grown on BAP with the four human 

blood types (A, B, AB, and O) (1).  

 

6

5

4,5

5

5,5

6

6,5

EDTA AB Defibrinated AB

T
h
e
 H

e
m

o
ly

si
s 

Z
o
n
e
 

A
v
e
ra

g
e
 (

m
m

)

Blood Type



 

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Figure 3. Hemolysis type of S. aureus on (a) BAP with EDTA-human AB blood type and (b) 

with defibrinated human AB blood type. 

 

S. aureus can produce the four types of 

hemolysis: α, β, γ, and δ. Certain S. aureus 

strains produce β toxin, which is a neutral 

sphingomyelinase (19). This toxin is 

produced in large quantities by some strains 

of S. aureus and secreted into culture media 

as an exotoxin with a molecular weights of 

35,000 (20).  

β-hemolysis is also referred to as 

complete hemolysis. β-hemolysin is highly 

influential on human immunity. Apart from 

hemolysin, there are other toxins that affect 

the virulence of S. aureus in humans. One of 

them is Panton-Valentine Leucocidin (PVL), 

which is a cytotoxin that is highly toxic to 

human neutrophils (21).  Certain strains of S. 

aureus also produce the toxic shock 

syndrome toxin-1 (TSST-1) which triggers 

toxic shock syndrome (TSS) (22). Other 

strains produce additional exoproteins, 

including TSST-1, staphylococcal 

enterotoxins (SEA, SEB, SECn, SED, SEE, 

SEG, SEH, and SEI), exfoliative toxins (ETA 

and ETB ), and leucocidin (20). 

 

Defibrination requires expertise and 

timeliness. An error may cause the blood to 

clot, leading to the imperfection of the media 

mixture and nutrients damage. EDTA could 

be used as a substitute for blood defibrination 

to grow S. aureus on BAP since it was able to 

have S. aureus produce the same type of 

hemolysis in defibrinated blood BAP, but 

with wider hemolytic zone and less colony 

growth. Furthermore, EDTA can simplify the 

process of preparing BAP as defibrination 

step was eliminated, thereby reducing the 

error factor in making BAP. 

 

CONCLUSIONS 

S. aureus could grow on BAP with 

EDTA-human AB blood type and 

defibrinated human AB blood type. EDTA 

could be used as a substitute for defibrinated 

blood in the process of making BAP to 

observe the hemolytic patterns of S. aureus, 

but could not be used as a benchmark for 

counting the bacterial colonies and 

measuring the hemolytic zones of S. aureus. 

a b 



 

 

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AUTHOR CONTRIBUTIONS 

Dora Dayu Rahma Turista: 

conceptualization, methodology, analyzing, 

writing-reviewing and editing. Eka 

Puspitasari: data curation, supervision, 

validation. Fanny Kurnanda: visualization, 

investigation, validation. 

 

 

ACKNOWLEDGMENT 

Gratitude was expressed to the 

Departement of Medical Laboratory (STIKes 

Hutama Abdi Husada Tulungagung, 

Indonesia) for the support in completing this 

study. 

 

CONFLICT OF INTEREST 

No conflict. 

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