MLT 415 -Lab Report (Antibiotic Sensitivity Test) PDF

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MLT 415 – Fundamentals of Microbiology Objectives: 1. To practice performing the antibiotic susceptibility testing following step-by-step procedure. 2. To observe the zones of inhibition and to measure the zones using metric ruler, callipers or a special transparent template. 3. To record the suscep...

Description

MLT 415 – Fundamentals of Microbiology

Objectives: 1. To practice performing the antibiotic susceptibility testing following step-by-step procedure. 2. To observe the zones of inhibition and to measure the zones using metric ruler, callipers or a special transparent template. 3. To record the susceptibility of the organisms to each antibiotic disk.

Principles: One of the important tasks of the clinical microbiology laboratory is to performance of antimicrobial susceptibility testing of significant bacterial isolates. The goals of testing are to detect possible drug resistance in common pathogens and to assure susceptibility to drugs of choice for particular infections. It is important to help the physician select effective antimicrobial agents for specific therapy of infectious diseases. The most widely used testing methods include broth microdilution or Kirby-Bauer method. Each method has strengths and weaknesses, including organisms that may be accurately tested by the method. Some methods

provide

quantitative

results

and

all

provide

qualitative

assessments. In general, current testing methods provide accurate detection of common antimicrobial resistance mechanisms. An antimicrobial is an agent that kills microorganisms or stops their growth. Antimicrobial medicines can be grouped according to the microorganisms they act primarily against. For example, antibiotics are used against bacteria and antifungals are used against fungi. They can also be classified according to their function. Agents that kill microbes are called microbicide, while those that merely inhibit their growth are called biostatic. The use of antimicrobial medicines to treat infection is known as antimicrobial chemotherapy, while the use of antimicrobial medicines to prevent infection is known as antimicrobial prophylaxis. Mechanisms of antibiotic action involve the blockage of a wide variety of metabolic activities such as energy metabolism, the function of bacterial membrane Page | 1

MLT 415 – Fundamentals of Microbiology

and the synthesis of peptidoglycan. The blockage of some of these functions is lethal to the cell growth. Susceptibility testing has shown that bacteria are becoming increasingly resistant to a wide variety of antimicrobial agents. Although new

antibiotics

continue

to

be

developed

by

pharmaceutical

manufacturers, the microbes seem to quickly find ways to avoid their effects. Two important bacteria that have developed resistance to multiple antimicrobial agents are Staphylococcus aureus strain, especially those resistant to the drug methicillin and its relatives and Enterococcus spp. resistant to vancomycin. AST results are often used to drive empiric antimicrobial therapy and formulary decisions in some cases are made based on AST results from the laboratory. It is commonly reported as the minimal inhibitory concentration (MIC), which is the lowest concentration of drug that inhibits the growth of the organism. Reports typically contain a quantitative result in µg/mL and a qualitative interpretation. The interpretation usually categorizes each result as susceptible (S), intermediate (I) or resistant (R). The disk diffusion technique (Kirby-Bauer method) historically has been and continues to be the method most commonly used for determining antibiotic susceptibility. In this technique, a fixed volume of nutrient broth containing a standard concentration of bacteria is smeared evenly onto the surface of an agar plate. Next, antimicrobial disks are applied to the plate surface. The culture plate is incubated, and as the bacteria grows on the surface of the plate, the antimicrobial diffuse out into the agar. After incubation, the plates are examined for the presence of zones of inhibition of bacterial growth (clear ring) around the antimicrobial disks as shown in figure 1. The zone of inhibition is measured in millimetres by using ruler and internal calliper. The edge of this zone correlates with the antibiotic concentration that inhibits the growth of the bacteria. The width of the zone is compared to a standard table. The size of the zone of inhibition Page | 2

MLT 415 – Fundamentals of Microbiology

depends on a number of factors, including the rate of diffusion of a given drug in the medium, the degree of susceptibility of the organism to the drug, the number of organisms inoculated on the plate, and their rate of growth. If the zone of inhibition is wider than the predetermined zone, the bacterial species is considered to be susceptible (S) to the antibiotic. If bacteria grow within the predetermined zone width, the species is considered resistant (R). In some cases, intermediate (I) designation is used if the zone of inhibition approximates the predetermined zone width. There are two possible reasons why the organisms tested was interpret in this categorize either the doses of drug are given to the patient is higher, or secondly, the infection is at a body site where the drug is concentrated. For example, the penicillin is excreted from the body by the kidneys and reaches higher concentrations in the urinary tract than in the bloodstream or tissues. When an interpretation of ‘intermediate’ is obtained, the physician may wish to select an alternative antimicrobial agent to which the infecting microorganisms is fully susceptible or additional tests may be necessary to assess the susceptibility of the organism more precisely.

Antimicrobial disk

Bacterial

Zone of inhibition

Figure 1: A disk diffusion antimicrobial susceptibility test. If the clear zones of growth inhibition around disks are of a certain diameter, the organism is susceptible to the antimicrobial agent in the disk.

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MLT 415 – Fundamentals of Microbiology

Table 1: Values listed in a standard chart (Adapted from Performance Standard for Antimicrobial Susceptibility Testing-National Committee of Clinical Laboratory Standard, NCCLS-2001)

Materials/Equipment: 1. Salmonella typhi, Salmonella paratyphi and Klebsiella pneumoniae Plate Culture 2. Tryptic Soy broth (TSB) 3. McFarland No 5 turbidity standard 4. Needles 5. Antimicrobial disks i. Ampicillin ii. Chloramphenicol iii. Tetracycline iv. Streptomycin 6. Wire Loop 7. Sterile Swab 8. Light Microscope 9. Meter ruler 10. Glass Slide 11. Culture Agar i. Nutrient Agar ii. Blood Agar iii. MacConkey Agar iv. Muller-Hinton agar 12. Immersion Oil 13. Normal Saline 14. Humidified Incubator 15. Electrical Incinerator 16. Gram stain reagent Page | 4

MLT 415 – Fundamentals of Microbiology

i. ii. iii. iv.

Primary stain (Crystal Violet) Mordant (Gram’s Iodine) Decolourizer (Acetone Alcohol) Counterstain (Safranin)

Procedures: A. Antimicrobial Sensitivity Test 1. A small colony of S.typhi, S.paratyphi and K.pneumoniae was transferred with sterilized inoculating loop into three different bottle of TSB. 2. The turbidity of each TSB was observed until approximately equivalent to that of the McFarland No. 5 turbidity standard. 3. Three Muller-Hinton agar plates were labelled properly. 4. The sterile swab was used to inoculate the bacterial from each of the TSB into the surface of Muller-Hinton agars plate respectively. 5. The needle was heated and different types of antimicrobial disks were placed on different region of the agars surface. 6. The agar plates were incubated upside down at 37°C for 24 hours. 7. The agar plates were removed from the incubator and the zones of inhibition in each of agar plates were observed and measured by ruler. 8. The susceptibility of each bacteria to each antibiotic disks were recorded as sensitive (S), resistant (R) or intermediate (I) following the guidelines included on the package insert of each disk cartridge. B. Inoculating of Culture Plate 1. The agar plates to be inoculated were selected and the bottom of each agar plates was labelled with a marker. 2. The wire loop was sterilized until reaching the red heat by using electrical incinerator. 3. A small portion of the S.typhi, S.paratyphi and K.pneumoniae was transferred into each agar plates. Page | 5

MLT 415 – Fundamentals of Microbiology

4. The lid of the agar plate was opened and the inoculum was spread over the one quadrant of the surface of the agar plate (A). 5. The wire loop was re-sterilized by using electrical incinerator until reaching the red heat. 6. The second quadrant of the plate was streaked by touching the end of first quadrant (B). 7. The wire loop was re-sterilized by using electrical incinerator until reaching the red heat. 8. The third quadrant was streaked by touching the end of second quadrant by making six to eight strokes (C). 9. The wire loop was re-sterilized by using electrical incinerator until reaching the red heat. 10. The fourth and fifth quadrant was streaked in a manner to produce isolate colonies (D and E). 11. The agar plates were placed upside down in the 37°C incubator to incubate overnight. 12. The agar plates were removed from the incubator and the growth of S.typhi, S.paratyphi and K.pneumoniae in the agar plates was observed. 13. The growth of S.typhi, S.paratyphi and K.pneumoniae colony morphology in each agar plates was recorded.

Figure 2: Diagram of plate streaking technique. The goal is to thin the numbers of bacteria growing in each of the plate as it is rotated and streaked so that isolated colonies will appear in sections D and E.

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MLT 415 – Fundamentals of Microbiology

C. Preparation of the smear 1. Two glass slides were prepared with the bottom side of each glass slide was labelled by drawing a circle using a marker. 2. The bacteria name was written on the frosted areas at each glass slide. 3. The wire loop was sterilized until reaching the red heat by using electrical incinerator. 4. A loopful of sterile normal saline was placed on each glass slide. 5. The wire loop was re-sterilized by using electrical incinerator until reaching the red heat. 6. A small portion of the each specimen from the culture plate was transferred into each glass slides. 7. The specimen was spread with an oval spiral movement by holding the wire loop in a flat position, move it outwards from the centre. 8. The wire loop was re-sterilized by using electrical incinerator until reaching the red heat. 9. The smear was dried by waving the glass slide in the air for a few minutes. D. Staining the smear 1. All the slides were placed on a staining rack and flooded the 2. 3. 4. 5.

slides with crystal violet for 1 minute. The crystal violet from the slides was wash with tap water. The slides were flooded with Gram’s iodine for 1 minute. The Gram’s iodine from the slides was washed with tap water. The slides were decolorized with acetone alcohol until the

slides appears colourless for 5 to 15 seconds. 6. The slides were briefly washed with tap water. 7. The slides were counterstained with safranin for about 1 minute. 8. The slides were briefly washed with tap water and blot dry. Page | 7

MLT 415 – Fundamentals of Microbiology

9. The slides were examined under the microscope and the outcome was recorded.

Results: A. Antimicrobial Sensitivity Test 1. Salmonella typhi

Antimicrobia l Agent

Concentra tion

Ampicillin Chloramphe

AMP10 C30

Zone Diameter (mm) 27 30

nicol Tetracycline Streptomyci

T10 S10

20 15

S.typhi (S, I or R) S S S S

n 2. Salmonella paratyphi

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MLT 415 – Fundamentals of Microbiology

Antimicrobial Agent

Concentrat ion

Ampicillin Chloramphen

AMP10 C30

Zone Diameter (mm) 27 28

icol Tetracycline Streptomycin

T10 S10

23 15

S.paratyph i (S, I or R) S S S S

3. Klebsiella pneumoniae

Antimicrobial Agent

Concentrat ion

Ampicillin Chloramphen

AMP10 C30

Zone Diameter (mm) 14 25

icol Tetracycline Streptomycin

T10 S10

20 19

K.pneumo niae (S, I or R) I S S S Page | 9

MLT 415 – Fundamentals of Microbiology

B. The Streaking & Morphology of Cultural Bacteria 1. The Nutrient Agar Culture Plate (Salmonella typhi)

Observati

1. Amount

ons: 2. 3. 4. 5. 6. 7. 8. 9.

of

growth:

Heavy

growth (3+) No of colony : Pure colony Shape : Circular Size : 0.5mm - 1mm Elevation : Flat Margin : Entire Colour : Cream Haemolysis : None Odour : Yes

2. The Blood Agar Culture Plate (Salmonella typhi)

Observati

1. Amount of growth: Heavy growth

ons: 2. 3. 4. 5. 6. 7. 8. 9.

(3+) No of colony : Pure colony Shape : Irregular Size : 1mm-3mm Elevation : Raised Margin : Undulated Colour : Grey Haemolysis : Gamma Haemolysis Odour : Yes

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MLT 415 – Fundamentals of Microbiology

3. The Nutrient Agar Culture Plate (Salmonella paratyphi)

Observati 1. Amount on: 2. 3. 4. 5. 6. 7. 8.

of

growth:

Moderate

growth (2+) No of colony : Pure colony Shape : Circular Size : 1mm -2mm Elevation : Flat Margin : Entire Colour : Cream Haemolysis : None

4. The Blood Agar Culture Plate (Salmonella paratyphi)

Observati 1. Amount on: 2. 3. 4. 5. 6. 7. 8.

of

growth:

Moderate

growth (2+) No of colony : Pure colony Shape : Circular Size : 2mm Elevation : Raised Margin : Entire Colour : Grey-white Haemolysis : Gamma Haemolysis

5. The Nutrient Agar Culture Plate (Klebsiella pneumoniae)

Observati 1. Amount

of

growth:

Moderate Page | 11

MLT 415 – Fundamentals of Microbiology

on: 2. 3. 4. 5. 6. 7. 8. 9.

growth (2+) No of colony : Pure colony Shape : Circular Size : 1mm Elevation : Flat Margin : Entire Colour : Cream Haemolysis : None Odour : Yes

6. The Blood Agar Culture Plate (Klebsiella pneumoniae)

Observati 10. on:

Amount of growth: Moderate

growth (2+) 11. No of colony : Pure colony 12. Shape : Irregular 13. Size : 1mm-3mm 14. Elevation : Raised 15. Margin : Undulated 16. Colour : Grey 17. Haemolysis : Gamma Haemolysis 18. Odour : Yes

7. The MacConkey Agar Culture Plate (Salmonella typhi)

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MLT 415 – Fundamentals of Microbiology

Observati on:

Non-Lactose Fermenter

8. The

MacConkey

Agar

Culture

Plate

(Salmonella

paratyphi)

Observati on:

Non-Lactose Fermenter

9. The

MacConkey

Agar

Culture

Plate

(Klebsiella

pneumoniae)

Observati on:

Lactose Fermenter

C. Staining the Bacteria 1. Salmonella typhi

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MLT 415 – Fundamentals of Microbiology

Observation: Gram negative bacilli

2. Salmonella paratyphi

Observation: Gram negative bacilli

3. Klebsiella pneumonia

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MLT 415 – Fundamentals of Microbiology

Observation: Gram negative coccobacilli

Discussion: In this experiment, three bacteria, Salmonella typhi, Salmonella paratyphi and Klebsiella pneumoniae were used to determine the susceptibility

these

bacteria

to

various

antimicrobial

agent.

This

standardized test is used to measure the effectiveness of a variety of antibiotics on a specific organism in order to prescribe the most suitable antibiotic therapy. In this experiment the Antimicrobial Susceptibility Testing (AST) method used was the disk diffusion method. This method is based on the inhibition of bacterial growth measured under standard conditions (standardization inoculum using McFarland standard). There are many AST technique developed by using the disk diffusion concept. The most widely used method is Kirby-Bauer which we used in this experiment. The production and use of the antibiotic penicillin in the early 1940s became the basis for the era of modern antimicrobial therapy and since then many other types of antimicrobials have been found and put into use. Thus, antimicrobial agents are used in the treatment of disease. In general the antimicrobial agent is an agent that kills microorganisms or Page | 15

MLT 415 – Fundamentals of Microbiology

stops their growth. The antimicrobial agent used in this experiment is Ampicillin,

Chloramphenicol,

Tetracycline

and

Streptomycin.

These

antimicrobial has different chemical properties which react differently. It can be any of a large variety of chemical compounds and physical agents as long it is used to destroy microorganisms or to prevent their development. Each of the bacteria was culture in separated Muller-Hinton agar plates. Each of the culture plate consist four antimicrobial disk located separately with one and another. Each of the culture plate was incubated at 37oC for 24 hours. All three culture plate was observed and the zone of inhibition was measured by ruler and recorded. The measurement obtained was compared with the value listed on standardize table provided. The first culture plate examined was the S.typhi culture plate. There are four visible inhibition zones with different diameter on the culture plate. Based on the measurement obtained, the inhibition zone for ampicillin is 27mm, the inhibition zone for chloramphenicol is 30mm, the inhibition zone for tetracycline is 20mm, and the inhibition zone for streptomycin is 15mm. Each of these reading was compare with the standard value listed on the table provided. These result can be interpret in three categories which either resistance, susceptible and intermediate. After a comparison between the value from the table and the value obtain from the measurement, it is indicate that bacteria S.typhi is susceptible with all microbial disk used in this experiment. The second culture plate examined was the S.paratyphi culture plate. There are also four inhibition zones with different diameter visible on the culture plate. Each of the inhibition zones was measured. The inhibition

zone

for

ampicillin

is

27mm,

the

inhibition

zone

for

chloramphenicol is 28mm, the inhibition zone for tetracycline is 23mm, and the inhibition zone for streptomycin is 15mm. After a comparison between the value from the table and the value obtain from the Page | 16

MLT 415 – Fundamentals of Microbiology

measurement, it is indicate that bacteria S.paratyphi is also susceptible with all microbial disk used in this experiment. The last culture plate examined was the K.pneumoniae culture plate. The inhibition zone for ampicillin is 14mm, the inhibition zone for chloramphenicol is 25mm, the inhibition zone for tetracycline is 20mm, and the inhibition zone for streptomycin is 19mm. After a comparison between the value from the table and the value obtain from the measurement, it is indicate that bacteria K.pneumoniae is susceptible with chloramphenicol, tetracycline and streptomycin while the ampicillin was categories as intermediate. Based on the result obtained, all the bacteria used in this experiment are either s...