Antibiotic Sensitivity Test Gentamicin in Bacteria Staphylococcus

aureus with Incubation Temperature 33ºC and 35ºC

Vincentia Ade Rizky

, Mei Rositasari

, Visensius Krisdianilo

, Julia Damayanti

and Sa’adah Siregar

Faculty of Pharmacy, Institut Kesehatan Medistra Lubuk Pakam,, North Sumatera, Indonesia

Faculty of Health Sciences, Universitas Katolik Musi Charitas, Palembang, Indonesia

Keywords: Sensitivity Test, antibiotic Gentamicin, Staphylococcus aureus

Abstract: Staphylococcus aureus is one of the bacteria that causes infection but is very resistant to various antibiotics

Many factors affect the results of this antibiotic sensitivity test, one of which is the temperature factor.

According to CLSI (2014) the incubation temperature for the sensitivity test used was 35 ± 2ºC, whereas

according to WHO (2004) the temperature used for this test was 35ºC temperature, temperatures higher than

35ºC of the culture seemed sensitive, whereas at temperatures lower than 35ºC resistant colonies will grow

inside the inhibition zone. This type of research is analytic observation. The research sample used was a pure

strain of Staphylococcus aureus ATCC 25923. The study sample was taken after fulfilling the inclusion and

exclusion criteria. Staphylococcus aureus bacteria that have been inoculated were planted on the Mueller

Hinton Agar media and antibiotics were added by the Kirby-Bauer method, then incubated at 33ºC and 35ºC

respectively. The inhibition zone yield is measured in mm. The measurement data then tested the hypothesis

by the Wilcoxon test with a 0.05 consecutive confidence level. The results showed the mean zone of inhibition

at 33ºC gentamicin 21.67 mm while at 35ºC gentamicin 21.67 mm. These data were analyzed and showed no

difference in the zone of inhibition of Staphylococcus aureus at 33°C and 35°C incubation temperatures.

Based on the results of the study it can be concluded that the difference in incubation temperature of 33ºC and

35ºC can be done in the examination of the Susceptibility Test because there is no difference between the

results of the zone of inhibition of Staphylococcus aureus bacteria with variations in incubation temperature.

1 INTRODUCTION

The bacteria Staphylococcus aureus is a bacterial

pathogen that can cause infections and disorders of

the skin include impetigo and folliculitis (Radji,

2010). The spread of Staphylococcus aureus bacteria

in the world is known as Methycillin Resistant

Staphylococcus aureus (MRSA). MRSA is an

infection of the bacterium Staphylococcus aureus

which is very resistant to various antibiotics that

causes new problems in the world of health. The last

overall report of the prevalence of Staphylococcus

and MRSA in Indonesia in 2006 reached 23.5%

(Ramadhani, 2014). Bacterial sensitivity test is a

method to determine the level of bacterial

susceptibility to antibacterial substances and to find

out the pure, antibacterial activity. The principle of

this method is inhibition of the growth of

microorganisms, which is a zone of obstacles will be

seen as a clear area around discs containing

antibacterial substances (antibiotics). The Diameter

of the bacterial growth barrier zone indicates bacterial

sensitivity to antibacterial substances. Furthermore,

the wider diameter of the zone of obstacles formed by

the bacteria is increasingly sensitive (Waluyo, 2008).

The sensitivity test result, as reported to the

Clinisi, is the classification of these microorganisms

into one of two or more sensitivity categories. The

simplest system consists of only two categories that

are sensitive and resistant. This classification has

many advantages for statistical and epidemiological

purposes, too rigid to be used by Clinilians.

Therefore, often used classification of three

categories. The Kirby-Bauer method and its

modifications are often used to determine the

sensitivity of the bacteria using three categories of

sensitivity, clinicians and laboratory workers must

understand the exact definition and clinical

significance of those categories (WHO, 2003). Many

factors affect the results of this antibiotic sensitivity

528

Ade Rizky, V., Rositasari, M., Krisdianilo, V., Damayanti, J. and Siregar, S.

Antibiotic Sensitivity Test Gentamicin in Bacteria Staphylococcus aureus with Incubation Temperature 33

C and 35

DOI: 10.5220/0009974305280535

In Proceedings of the International Conference on Health Informatics and Medical Application Technology (ICHIMAT 2019), pages 528-535

ISBN: 978-989-758-460-2

test, including environmental factors. Environmental

factors that affect growth include abiotic and biotic

factors. Abiotic factors include temperature, osmose

pressure, drying, and ions from electricity. Biotic

factors are among the factors found in the bacteria

itself (Gultom, 2015). Temperature is the most

important factor in influencing the rate of bacterial

growth. Sometimes temperature is not much noticed

in growth and of bacterial identification. Laying

bacterial isolation at room temperature when

incubators are being used for the safety of other

bacteria often done. Temperature analytic procedures

should be observed to diagnosis in the breeding of

bacteria is acceptable. Influence incubation

temperature may result in an error analytic procedure

to fault inhibition zone results (Safety, 2015).

The optimum growth temperature for

Staphylococcus aureus is 35-37ºC. This sensitivity

test was incubated at 35ºC for optimal growth. If the

temperature is lowered, the time needed for effective

growth will lengthen and produce a wider zone. At

temperatures of 35ºC or lower, colonies can grow

inside the inhibition zone. The incubation time is 16-

18 hours (for rapid diagnosis), but you should use a

conventional 24 hour time for optimum results

(Vandepitte et al., 2010). The bacterial temperature

metabolism Stapylococcus aureus is when the

temperature rises, the metabolic rate in the form of

macromolecules such as proteins, nucleic acids, in

Stapylococcus aureus bacteria will suffer damage

permanent (Abrar et al., 2013). When lowered, the

metabolic rate descending (Brooks et al., 2005).

According to Madigan et al (2012) the bacterial

growth temperature can be divided into psychophilic

bacteria (15ºC-20ºC), mesophilic bacteria (20ºC-

40ºC) and thermophilic bacteria (50ºC-60ºC).

Staphylococcus aureus bacteria whose growth is

optimal in temperatures of 35-37ºC are included in

mesophilic bacteria (Vandepitte et al., 2010).

According to WHO on Basic Laboratory the

incubation temperature used for the optimal

sensitivity test is 35ºC with a time of 16-18 hours or

24 hours

(WHO, 2003)

. CLSI on Performance Standards

for Antimicrobial Suceptibility suggests using a

temperature of 35 ± 2ºC (CLSI, 2014). Previous

research stated that temperatures of 37ºC and 40ºC

were effective for the growth of Staphylococcus

aureus toxins and enterotoxins (Vandenbosch et al.,

1973). Other studies on the isolation and

characterization of Staphylococcus aureus of Milk of

Ettawa Crossbred Goat, were inoculated on the

Mueller Hinton media in temperature 37 ºC to obtain

intermediet results (Purnomo, 2006). Antibiotic

sensitivity test according to CLSI uses a temperature

of 35 ± 2ºC (33ºC and 37ºC). According to WHO on

Basic Laboratory the temperature used is 35ºC, at

temperatures higher than 35ºC the culture appears

sensitive, whereas at temperatures lower than 35ºC

the resistant colony will grow inside the inhibition

zone (WHO, 2003).

2 MATERIAL AND METHODS

The study was conducted at the Microbiology

Laboratory at Grandmed Lubuk Pakam Hospital. This

Research uses the Wulcoxon Test statistical analysis.

In this research the tools and materials used include:

Ose, Autoclave, Bunsen, sterile cotton swab, test

tube, ruler/ calipers, Nutrient Agar, Mueller Hinton

Agar, Antibiotic Gentamicin, NaCl 0.9% sterile,

Incubator. Media MRVP, Media Lactose, Media

Maltose, H

3%, Coagulase Test, DHO, Mac-

Farland 0,5, Gentian Violet, Lugol/Iodium, Alkohol

96%, and Safranin.

The first step in this research is sterilize the tools

and materials. The tools used must be sterile. The tool

is sterilized using a Dry Heat Oven (DHO) at a

temperature of 160ºC for 2 hours. Then, the materials

used for testing and bacterial growth after being made

in accordance with the specified composition are then

sterilized using an autoclave at 121ºC for 15 minutes.

The next step is to do sterility and quality test of the

media. Steps to do the media sterility test is put the

media Mueller Hinton Agar, Nutrient Agar, Lactose,

Maltose to the incubator at 35ºC for 18-24 hours.

Then proceed witd testing the quality of the media is

create Staphylococcus aureus bacterial suspense with

Mac Farland turbidity level 0.5, the do 10.000

dilution suspensions. Then obtained 10

dilutions or

equivalent to the number of colonies from 100-200.

The media to be tested is 10% of the amount of media.

After obtaining the media to be tested then take 100

µL suspense that has been diluted then inoculated in

the media using the spread plate method. Incubate for

18-24 hours at 35ºC. The next steps is making the

suspension bacteria, take 1 ose of bacterial suspense.

Scratch the Nutrient Agar media evenly with the

Streak method. Incubate for 18-24 hours at 35ºC.

2.1 Bacteria Standard Test

Catalase Test. Procedure catalase test (Figure 1) is

Apply one drop of H

3% on a object glass, then

transfer the Staphylococcus aureus bacterial colony

with a loop to the H

solution, then mix. Catalase

positif reaction:evident by immediate effervescence

(air bubble formation), and then catalase negatif

Antibiotic Sensitivity Test Gentamicin in Bacteria Staphylococcus aureus with Incubation Temperature 33

C and 35

529

reaction: no bubble formation (no catalase enzyme to

hydrolyze the hidrogen peroxide. The catalase test is

primarily used to distinguish among gram positive

cocci: members of the genus Staphylococcus are

catalase positif, and members of genera Steptococcus

and Enterococcus are catalase negatif (Tankeshwar,

2013).



Figure 1: Catalase Test (Vetbatch, 2017)

Microscopic Test. Make bacterial suspense from

bacterial colonies that grow on agar nutrient media.

Make preparations slide of bacteria, then do gram

staining. The steps of gram staining (Figure 2) : (a)

fixation of the slide, (b) apply a crystal violet stain for

1 minute, (b) wash slide in a gentle and indirect

stream of tap water for 2 seconds, (c) flood slide with

the iodine wait 1 minute, (d) wash slide in a gentle

and indirect stream of tap water for 2 second, (e) flood

slide with decolorizing agent (alcohol decolorizer)

wait 10-15 second or add drop by drop to slide until

decolorizing agent running from the slide runs clear,

(f) flood slide with a counterstain with safranin, wait

30 second to 1 minute, (g) wash slide in a gentile and

indirect stream of tap water until no color appears in

the effluent and then blot dry with absorbent paper,

(h) observe the resulth of the staining procedure under

oil immersion using a microscope (Tankeshwar,

2015). The result of gram staining is gram negatif

bacteria will stain pink/red and gram positif bacteria

wil stain blue/purple.

Figure 2: Gram Staining (Prajapati et al., 2018)

The next step of bacteria standart test is

biochemical characterization. Potential isolate were

characterized by different biochemical methods like

lactose and maltose test, MR (Methyl red) and VP

(Voges Proskauer) test, and Coagulase test. Sugar

Test (Lactose and Maltose) : Take 1 ose of bacterial

suspense and put it in a sugar medium (lactose and

maltose). Mix evenly. Incubate at 35ºC for 18-24

hours. Methyl Red (MR) Test : Take 1 ose bacterial

suspense, insert into MR media, mix evenly, incubate

at 35ºC for 18-24 hours. After incubating to find out

the reaction, add the Methyl red reagent. Positive

results are indicated by a change in color from yellow

to red. Voges Proskauer Test (VP) :

Take 1 ose

bacterial suspense, insert into the VP media, mix

evenly, incubate at 35ºC for 18-24 hours. After

incubating to find out the reaction, add the KOH and

α-Naphthol reagents. Positive results are indicated by

a change in color from yellow to red. Coagulase Test

: This test is used to determine the presence of free

coagulase by means of 200 µL plasma aseptically

inserted into a sterile test tube. Take 3-5 bacterial

colonies of Staphylococcus aureus , put them in the

test tube, mix carefully. Incubate at 35ºC for 18-24

hours.

The next step is prosedure of antibiotic

sensitivity test. The first Making the Inoculum

(Suspension) : Take 3-5 ose Staphylococcus aureus

bacteri colonies of the same size in the media using

an ose (loop) that has been incanded above Bunsen.

Then insert the colony into a test tube that contains

sterile 0.9% NaCl solution, then homogeneous.

Compare this with turbidity suspension of

Staphylococcus aureus with 0.5 Mac-Farland

comparison solution. NB: Turbidity Bacterial

suspension should be equivalent to Mac-Farland 0.5.

And then, step of Antibiotic Sensitivity Test

(Susceptibility Test) : Inoculation using the Kirby-

bauer method and place the antibiotic in the middle of

the media surface. Incubate the media at 33ºC and

35ºC for 18-24 hours. And then, record and measure

each zone formed around the antibiotic (disc paper).

The resulting inhibition is measured in mm

(millimeters), show procedure in Figure 3.

ICHIMAT 2019 - International Conference on Health Informatics and Medical Application Technology

530

3 RESULT

Identification of Staphylococcus aureus bacteria can

be microscopic and biochemical test of bacteria. On

microscopic examination found Gram (+) cocci (+).

These bacteria are round like a ball or spherical,

clustered and purple. The results obtained are the

same as the results of microscopic examination by

Holt et al (1994) which states that the Staphylococcus

aureus bacteria are gram-positive coccus bacteria that

are spherical in shape like a ball. These bacteria are

purple because it is the first absorbing dye is gentian

violet. The result of microscopic test show in Figure

4 below.

Figure 3: Step Antibiotic Test (ACS, 2020)

Figure 4: Microscopic Test

Biochemical tests used to test further

identification of Staphylococcus aureus bacteria. This

test is carried out on confectionery media namely

lactose and maltose, Methyl Red (MR), Voges

Proskauer (VP), Catalase Test and Coagulase Test.

The results can be seen in Table 1 and Figure 5. The

Result of Staphylococcus aureus bacterial

biochemistry test carried out in confectionery media,

namely lactose and maltose, showed positive results

with a marked change in green to yellow, indicating

that the bacteria were able to ferment lactose and

maltose. Patty research states that the test of lactose

and maltose sugars of the bacterium Staphylococcus

aureus get positive results that are marked by a

yellow discoloration, this shows that the bacteria

tested are able to ferment the type of sugars tested

(Patty et al., 2016).

In the Methyl Red and Voges Proskauer tests,

positive results were obtained with a change from the

deep red yellow which indicates the presence of

acetoin in the solution produced by bacteria. Dewi’s

research states that the positive results of the Methyl

Red and Voges Proskauer tests are characterized by a

change in color from yellow to red indicating the

presence of glucose fermentation in Staphylococcus

aureus from the formation of acetilmetilcarbinol

(acetoin) (Dewi, 2013). Catalase test is a test to

distinguish between groups of Staphylococcus aureus

or Steptococcus bacteria. The results obtained in the

catalase test are positive which is marked by the

presence of air bubbles (O

). Toelle’s research states

that the Staphylococcus aureus bacterial catalase test

with positive results because these bacteria produce

enzymes capable of hydrolyzing hydrogen peroxide

) into water (H

O) and air bubbles (O

) (Toelle

& Viktor, 2014). On the examination of the coagulase

test positive results were indicated by the presence of

lumpy white jelly. Purnomo’s research states that

Staphylococcus aureus can agglutinate blood,

bacause it has a coagulase reacting factor, the role of

coagulase produced by germs is used as a determinant

of Stapylococcus aureus species that make white clots

like jelly that show positive results (Purnomo, 2006).

Table 1: Test Results for Identification of Staphylococcus

aureus Bacteria



Figure 5: Biochemical Test

Identification Test Results

Lactose Test (+) Positive yellow

Maltose Test (+) Positive yellow

Metyl Red Test (+) Positive red color

Voges Proskauer (+) Positive red color ring

Catalase Test (+) Positive air bubbles

Antibiotic Sensitivity Test Gentamicin in Bacteria Staphylococcus aureus with Incubation Temperature 33

C and 35

531

Sterility test in this study was carried out on Nutrient

Agar Media and Muller Hinton Agar Media. This test

is performed by non-cultivation of Staphylococcus

aureus. The media standard will be tested by means

incubated at 37

C for 24 hours. The purpose of the

media sterility test is to determine whether there is

growth of bacteria and fungi of other microorganisms

that can affect the results of research. The results of

sterility media test can be seen in Table 2 and Figure 6.

Table 2: Media Sterility Test

Media Test result Sterile / Not steril

Nutrient Agar Does not

grow

Sterile

Mueller Hinton

Agar

Does not

grow

Sterile

Figure 6: Nutrient Agar Media and Mueller Hinton Agar

Media for media sterility test result

Media quality test performed to determine the growth

of bacteria on a medium which will be used, whether

the media can be overgrown with fertile bacteria or

not. This test is performed on Nutrient Agar and

Mueller Hinton Agar. The results can be seen in Table

3, Figure 7 and Figure 8.

Table 3: Media Fertility Test

Media Test result

(Colonies)

Good/

Not Good

Nutrient Agar Grow Good

Mueller Hinton

Agar

Grow Good

Figure 7: The results of the agar nutrient media quality test

Figure 8: The results of the agar Mueller hinton media

quality test (Mueller Hinton Agar with Gentamicin

Antibiotic Media is shown in the image on the left

3.1 Results of Gentamicin Antibiotic

Sensitivity Test (Susceptibility Test)

Susceptibility test or antibiotic sensitivitty test is used

to determine the inhibitory zone produced from an

antibiotic. Inhibition zone was used as the diagnosis

result if the antibiotics are sensitive or resistant to

these bacteria. The results of the Staphylococcus

aureus bacteria inhibition zone on Gentamicin

antibiotics with an incubation temperature of 33ºC

(Figure 9) and 35ºC (Figure 10) for 24 hours are

presented in Table 4.

Table 4: Results of inhibition zones of Staphylococcus

aureus bacteria on Gentamicin antibiotics with an

incubation temperature of 33ºC and 35ºC.

Gentamicin Antibiotic Zone

(mm)

Information

Temperature

of 33ºC

Temperature

of 35ºC

1 21 22 Sensitive

2 22 22 Sensitive

3 21 21 Sensitive

4 21 22 Sensitive

5 22 22 Sensitive

6 21 21 Sensitive

7 22 22 Sensitive

8 22 22 Sensitive

9 23 21 Sensitive

Average 21.67

21.67

Sensitive

Description :

Sensitive ≥ 15mm; Resistance <12mm

ICHIMAT 2019 - International Conference on Health Informatics and Medical Application Technology

532

Figure 9: The result of inhibition zone of antibiotic

gentamicin at a temperature 33ºC

Figure 10: The result of inhibition zone of antibiotic

gentamicin at a temperature 35ºC

3.2 Data Analysis

The results of the examination of the zone of

gentamicin antibiotic inhibition of bacteria

Staphylococcus aureus that have been collected, then

processed and then presented in tabular form and

analyzed the data through several stages, namely

normality test and hypothesis testing.

3.2.1 Normality Test

In this study the normality test was carried out using

the Kolomogorov-Smirnov test because the number

of samples used was less than 50 with a significance

level of 95% (=0.05). The following are normality

test results presented in Table 5 below:

Table 5: Normality Test Results

Kolomogorov-Smirnov

Statistic Df Sig

Temp33 ,272 9 ,054

Temp35 ,414 9 ,000

Based on Table 5 the p value (sig) obtained for the

temperature of 33ºC gentamicin antibiotic is 0.054,

for temperature 35ºC gentamicin antibiotic is 0,000.

From the values above there is 1 data that is normally

distributed at 33ºC, while at 35ºC the data is not

normally distributed. Because there is one data that is

not normally distributed, it is followed by a non-

parametric statistical test.

3.2.2 Hypothesis Testing

The Wilcoxon Test is a test used to test differences in

sample pairs in 2 groups (Dahlan, 2014). The results

of data analysis are obtained as shown in Table 6.

Table 6: Wilxocon Test Results

Temp 33 -

Temp 35

,000

Asymp. Sig. 1,000

From the analysis of the data obtained, it can be seen

that the significant value in the Table above is 0,000.

Based on the specified conditions if it is significantly

greater than the alpha value of 0.05 then Ho is

accepted, whereas if it is significantly smaller than the

alpha value of 0.05, Ho is rejected. So it can be seen

that the asymp.sig value obtained is 1,000 is greater

than the alpha value of 0.05 then Ho is accepted.

Thus, it can be concluded that "There is no difference

in the inhibition zone in the Gentamicin Antibiotic

Sensitivity Test for Staphylococcus a ureus Bacteria

for Incubation Temperature Variations of 33ºC and

35ºC".

4 DISCUSSION

In this study, a number of preliminary tests were

carried out including sterility tests, quality tests, and

bacterial standard tests. Sterility tests are carried out

with the aim of ensuring that the media used are not

contaminated with bacteria or other microorganisms.

Fertility test or media quality test is a test used to see

that the media used is good for bacterial growth.

Bacterial standard tests are also used to identify or

confirm tests that the bacteria used are

Staphylococcus aureus bacteria.

Identification of Staphylococcus aureus bacteria

can be done with microscopic tests and bacterial

biochemical tests. On microscopic examination, gram

(+) coccus (+) was obtained. These bacteria are round

like a ball or spherical, clustered and purple. The

results obtained are the same as the results of

microscopic examination by which states that the

Staphylococcus aureus bacteria are gram-positive

positive coccus bacteria that are spherical in shape

like a ball. This bacterium is purple because it absorbs

the first dye, gentian violet (Holt et al., 1994).

Staphylococcus aureus bacterial biochemistry test

carried out on confectionery media, namely lactose

and maltose, showed positive results with a marked

change in green to yellow, indicating that the bacteria

Antibiotic Sensitivity Test Gentamicin in Bacteria Staphylococcus aureus with Incubation Temperature 33

C and 35

533

were able to ferment lactose and maltose. In the

Methyl Red and Voges Proskauer tests positive results

were obtained with a change in color from yellow to

red mangosteen which showed the presence of

acetoin in the solution produced by bacteria. Catalase

test is a test to distinguish between Staphylococcus

and Streptococcus bacteria groups , in this test

positive results are indicated by marked bubbles when

dripped with H

In the examination of the

coagulase test obtained positive results in the

presence of lumpy white jelly. The results obtained

are the same as the results of Dewi's research (Dewi,

2013).

In this study the results of gentamicin antibiotic

inhibition zones, each incubated at 33

C and 35

showed no difference because the temperature range

used in the growth temperature range of

Staphylococcus aureus bacteria was either used or

commonly called mesophilic temperature. According

to the research of James H. Jorgensen et al the

optimum growth of Staphylococcus aureus at 35

for 16-24 hours with vancomysin, daptomycin, and

linezolid antibiotics (James et al., 2009). According

to the Clinical and Laboratory Standars Institute

(2014) sensitivity test or antimicrobial susceptibility

test using temperatures of 35 ± 2ºC (33ºC, 35ºC and

37ºC). Meanwhile, according to WHO on Basic

Laboratory, it is recommended that the temperature

sufficiency test be used for 35 ° C for 24 hours,

because at the most optimal temperature it can reduce

the risk of contamination of other bacteria that will

grow (WHO, 2003).

Analysis of the data used in this study is

Wilxocon. Based on Wilxocon test results, it is

known that there is no difference in the zone of

inhibition of Staphylococcus aureus bacteria on

gentamicin antibiotics with incubation temperatures

of 33ºC and 35ºC. The results obtained are seen from

the value of sig. 1,000 is greater than the alpha value

(1,000> 0.05).

5 CONCLUSION

Based on the results and discussion carried out it can

be concluded as follows;

a. The average zone of inhibition of the antibiotic test

for gentamicin in Staphylococcus aureus with an

incubation temperature of 33ºC was 21.67 mm

b. The average zone of inhibition of the antibiotic test

for gentamicin in Staphylococcus aureus with an

incubation temperature of 35ºC was 21.67 mm

c. From the results of gentamicin antibiotic inhibition

zones that have been carried out statistical tests, it

can be concluded that there is no difference in the

zone of inhibition in the antibiotic sensitivity test of

Staphylococcus aureus bacteria to the incubation

temperature variations of 33ºC and 35ºC.

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