Gram-Staining, Catalase Test and Oxidase Tests

Introduction

There is a cutting-edge world enlarge in drug-resistant human pathogens inclusive of bacteria, fungal and viral pathogens which poses a serious risk to health-care. Hence, microbiology is fundamental for find out about by means of pharmacists for motives such as: infectious ailment diagnosis, cure and prevention, discovery of vaccines and different antimicrobial drugs, and to make certain that drug healing procedures utilized goal opportunistic microbes except harming the host. The present day climatic modifications round the world and problems such as air pollution have made it beneficial for a number of microbial organisms to multiply and advance resistance to medication. Further, doubling time in microbiology estimates transmission potential. High doubling time lead to greater transmission of infections and different influences such as higher meals spoilage and vice versa. This in addition proves the significance of microbiology in tackling such challenges and advertising well-being which is the intention of pharmacists. Microbial identification involves the use of two main groups: genotypic and phenotypic identification. Genotypic deals with an organisms full hereditary information whereas the phenotypic deals with organisms observed characteristics such as morphology and behavior. Phenotypic methods are mainly used in the laboratory due to their lower costs. The techniques applied in the laboratory include streak plate, pour plate method, spread plating, replica plating and soft-agar overlay techniques.

The experiment performed was a case of a patient-Mr J. Virgo aged 63, has undergo a recent surgical procedure hernia repair, and has developed a severe infection presenting as shock and possible organ failure. After admittance to the hospital Mr J. Virgo was diagnosed as presenting-shock syndrome. The aim of this experiment was to test samples taken from Mr J. Virgo to identify the microorganisms which can cause the toxic-shock syndrome. The microorganisms tested for in the laboratory include Streptococcus, Escherichia, Lactobacillus and Serratia.

Method

Gram Staining

The gram staining technique involved four necessary procedures. We applied a crystal violet stain (primary stain) and the methyl violet to a heat-fixed smear of bacterial culture. This was followed by the addition of Gram iodine, the mordant. The third step was rapid decolourization with alcohol and acetone and counterstaining with basic fuchsin.

The slide by placing the bacteria on the slide with a drop of water. Then allowed them to dry and heat-fixed it. A monolayer of organism sufficiently dense for easy visualization but thins enough to reveal typical morphological characteristics were observed. The experiment used the same pipette or swab, and inoculated culture media first. We fixed the smear using methanol, which helped prevent the distortion of cells. This was helpful in the microscopic observation of CSF and urine. Alternatively, you can make multiple smears, which is economical and saves on time.

On the rack, we flooded the crystal violet( methyl violet) and then briefly washed to remove excess crystal violet. We then flooded the gram stain for ten seconds and quickly soaked in water to let the section dry out. The acetone was then decolorized for a few seconds until the moving dye front has passed the lower edge section. We immediately washed in tap water and counterstained with Safranin for 15 seconds. Note that the most critical step in gram staining is decolorization step since the crystal violet stain will be removed from both G positive and G negative cells if the decolourizing agent is left too long. Acetone is a better decolorizer than alcohol and decolourizes more rapidly; therefore, it must be used with a lot of care. Exessive decolorization turn gram-positive appear gram-negative. Gram staining depends on the culture age, staining method, and incubation atmosphere. Similar consideration is also applied in the interpretation of smears from clinical specimens. Additional factors include different host cell types and possible phagocytosis. Gram stain allows the separation of all bacterial into two broad groups. Gram stain works empirically since we know that gram reaction is based on the structure of the bacterial cell wall. In Gram-positive bacteria, the dark purple crystal violet stain is retained by the thick layer of peptidoglycan, which forms the outer layer of the cell. In Gram-negative bacteria, the thin peptidoglycan layer in periplasm does not keep the dark stain, and the pink Safranin counterstains the peptidoglycan layer.

Catalase

A small amount of bacterial colony on a clean glass slide. We then added one to two drops of 3% hydrogen peroxide. The presence of rapid bubble formation indicates positive results, while no bubble formation suggests negative consequences. Hydrogen peroxide was directly added to the culture on the microscopic slide. A positive result by staphylococcus aureus was indicated by bubbling, whereas the lack of bubbling means adverse reactions produced by streptococcus pyogenes. Staphylococcus aureus uses catalase to neutralize hydrogen peroxide.

Oxidase Test

The study used filter paper soaked with the substrate tetramethyl p phenylenediamine dihydrochloride. We then moistened the paper with distilled water and chose the specific colony to be tested with a wooden or platinum loop. We then smeared on the filter paper. Finally, we observed the inoculated area of writing for the color change to deep blue or purple within ten to thirty minutes. Alternatively, you can take a commercially available oxidase disc which contains the reagent. You first pick the isolated colony to be tested and rub in the drive. Finally, observe the color change with the first ten minutes. The development of a deep purple color will indicate a positive test, while the absence of any color will show a negative test.

Results

Time (mins)

CFU/ml Repetition 1

CFU/ml Repetition 2

CFU/ml Repetition 3

CFU/ml Repetition 4

0

2800000

27000000

22000000

20000000

30

2120000

25100000

22000000

25000000

60

2180000

24200000

23100000

22000000

90

3500000

27000000

29000000

13600000

120

3600000

31000000

34000000

30000000

150

4000000

41000000

39000000

37000000

180

4600000

43000000

47000000

47000000

210

7900000

77000000

71000000

74000000

240

11300000

12100000

115000000

11000000

270

25000000

22000000

275000000

24000000

300

27000000

26000000

290000000

22000000

330

31000000

29000000

215000000

21000000

360

32000000

300000000

350000000

37000000

SPECIES IDENTIFICATION

Tested Organism

Streptococcus agalactiae

Staphylococcus aureus

Streptococcus pyogenes

Enterococcus faecalis

Mannitol utilisation

–

–

+

–

+

Haemolysis( On blood sugar plate)

Beta

Usually beta

Usually beta

Usually beta

Usually gamma

Colony Pigmentation (blood agar plate)

cream

Usually cream

Usually golden

Usually cream

Usually cream

DNA hydrolysis

+

–

+

+

–

Bacitracin sensitivity (s) or Resistance ®

S

R

R

S

S

Coagulase Test

–

–

+

–

–

The identified species was further identified examined using bacterial enumeration techniques in Nutrient Broth at 37 degrees celcius

Spectrophotometry Results

Time(mins)

ABS @ Repetition 1

ABS @ Repetition 2

ABS @ Repetition 3

ABS @ Repetition 4

0

0.04

0.04

0.037

0.032

30

0.044

0.067

0.055

0.042

60

0.05

0.065

0.069

0.058

90

0.07

0.11

0.09

0.099

120

0.134

0.114

0.121

0.131

150

0.286

0.284

0.302

0.267

180

0.518

0.497

0.510

0.509

210

0.634

0.612

0.641

0.633

240

0.891

0.843

0.793

0.823

270

1.107

0.998

1.052

1.034

300

1.233

1.333

1.273

1.283

330

1.334

1.439

1.339

1.411

360

1.435

1.419

1.406

1.519

Mr J aged 63 has undergone a recent surgical procedure, hemia repair and developed seven infections presenting as shock and possible organ failure. AAfter admittance to the hospital Mr Virgo was diagnosed as presenting with toxic shock syndrome. Samples have been taken by the attending physician and have been sent to microbiology fro testing. Several bacteria and vruses can cause toxic shock syndrome and therefore diagnosis and specific treatment required

All raw data was acquired using the same protocols utilized in the lab section

Description of colony characteristics

Growth Characteristics

NaCl

Bile

Azide

Polymyxin

Control

Colony type 1

Transparent, small and flat

–

–

–

+

++

Colony type 2

Red, small and raised

–

++

–

–

+++

Colony type 3

White large and round

+++

++

++

+

+++

Colony type 4

Cream, large and round

–

+

–

–

+++

Gram staining, Catalase and Oxidase Test were performed on four isolated bacterial slide

Colony type

Gram-reaction

Shape

Catalase

Oxidase

1

+

Coccus,chains

–

–

2

–

Coccus

+

–

3

+

Rod

–

–

4

–

Rod, Clusters

+

–

DiscussionStaphylococcus aureus has been identified as the micro-organism causing acute cellulitis infection in Mrs. Hendry’s case using the different experimental techniques of microbiology. Initially, the results obtained from the mixed selective media used. Table 1 provides a brief identity of the micro-organism. (5)DT, Petrico, 1973). Firstly the particular medium NaCl selects for halotolerant bacteria. As a result, the bacterium present in Mrs. Hendry’s skin swaps should e tolerant because both AS and BS had good growth on the NaCl media. Secondly, polymyxin inhibited the growth of some samples, but the sample AS BL had good growth, and BS had limited growth. Polymyxin allows gram-negative bacteria to grow as it does not affect its outer membrane. Gram staining reactions differentiates between gram-negative and gram-positive bacteria. Both bacteria have different cell wall structures. Gram-positive bacteria have a thick, dense crosslinked layer of peptidoglycan that forms a cell wall. Because of this methyl violet color is retained with its bacterial cell wall after counterstaining with Safranin takes place. Therefore when observed under the microscope, the cultures appear blue or purple if they are gram-positive. This was seen on each of the AS and BS microscopic slides. In Gram harmful bacteria, the peptidoglycan is thinner and non- crosslinked layer forming the cell wall. As a result, the methyl violet is removed when the dye is decolorized using acetone and counterstained with Safranin. So, gram-negative bacteria appear red or pink.

Methyl violet is removed when the dye is decolorized using acetone and counterstained with Safranin. So gram-negative bacteria appear red or pink when observed under the microscope. This is found in the AL sample in table 2. Gram-positive bacteria form spores. BL sample appeared to be pink and purple pigmentation under the microscope. This suggests that the bacteria in the BL sample contained spores, which are oval-like structures growing within the gram-positive bacterial cell wall. BL sample appeared to have pink and purple pigmentation under the microscopic under the microscope. This suggests that the bacteria in the BL sample contained spores which are oval-like structures growing within the bacterial cell wall due to dehydration of the cytoplasm. The spores have thinner peptidoglycan wall. Therefore, gram staining results appeared pink as in gram-negative bacteria. However, a purple-stained cell within the BL culture represents gram-positive bacteria because of the reason which have been explained above.

A further test, for example, catalase and oxidase tests were carried out to gain more information about the samples. The catalase test showed which bacteria in samples tested showed tested produced cytochrome c oxidase. This is an enzyme found in the electron transport chain (ETC) present in the mitochondria. When this enzyme is present, the colorless oxidase reagent (Tetramethyl – p- phenylenediamine) is used in the oxidation of indophenols of purple color. This is seen for each of BS and BL samples, whereas each of AS and AL were oxidized negative as oxidase reagent remained colorless, suggesting cytochrome c oxidase was not present in these samples. In table 2, similarly, the catalase test differentiates the catalase containing bacteria. Examples of AS, AL, BS, and BL produced gas bubbles when added to catalase reagent( Hydrogen peroxide). (6)This showed that the hydrogen peroxide was broken down into water and oxygen, which are less harmful by the catalase enzyme. Catalase enzyme was present in the bacterial sample. Using these test, the genus of the four samples were identified. Colony type was staphylococcus, AS. Staphylococcus caused cellulitis infection and was responsible for causing acute cellulitis infection in Mrs. Henry. In the identification of species Staphylococcus, differential media was used. For example, mannitol was fermented by staphylococcus, and differential media was used. For example, mannitol was fermented by the staphylococcus, and a differential media was employed. The clour changed from red to yellow as the pH was reduced. In addition to that the blood medium presented beta hemolysis and sensitivity to novobiocin. This suggested that the novobiocin compete in the inhibition of the DNA gyrase, therefore, blocked the relaxation of super-coiled bacterial DNA.

As a result, the transcription and translation process of DNA was blocked hence binary fission, and this leads to the eventual death of the bacteria. Also, staphylococcus was grown in the DNA medium. Positive results were obtained, and there was decolorization of the methyl green in the medium. Table 3 suggests that deoxyribonuclease (DNase) was produced by the staphylococcus grown. DNase is an enzyme that degrades DNA by hydrolysis of the phosphodiester bond, resulting in the break down of the double helix strand. (7) The medium changes from methyl green to clear color because methyl green binds to the DNA back backbone. Upon the breakage of this, the methyl green is no longer attached to anything resulting in a change of tone. Collectively the mannitol blood and DNase test were found compatible with results from the previous studies proving that staphylococcus species was present in Mr. Henry’s skin swap was S. aureus. According to Lambert-Beer law, the absorption was directly proportional to the concentration of the substrate and the path of the length of light through the solution.

Conclusion

Bacteria are stained with crystal violet stain, and all the bacteria will be stained purple color. Gram Iodine solution is added to the solution, and the stained will be fixed due to the formation of sophisticated crystal violet and iodine. Add a decolorizer, for example, alcohol or acetone. Some cells will decolorize while some cells will not decolorize. i.e. they will retain the stain. Use a counterstain; for instance, Safranin for the final process of the procedure and those cells that will keep the primary color of stain are Gram-Positive. (8) Those that do not retain the color of the stain and take up the color of the counterstain are called Gram-Negative Bacteria.

The enzyme catalase mediates the breakdown of hydrogen peroxide into oxygen and water. The presence of the enzyme in a bacterial isolate is evident when a small inoculum is introduced into hydrogen peroxide, and the rapid elaboration of oxygen bubbles occurs. The lack of catalase is apparent by a lack of or weak bubble production. The culture should not be more than 24 hours old. Bacteria thereby protect themselves from the lethal effect of Hydrogen peroxide, which is accumulated as an end product of aerobic carbohydrate metabolism.

The ability of an organism to produce the cytochrome C oxidase can be determined by using the reagent tetramethyl-p-phenylenediamine dihydrochloride impregnated in the filter disk. The reagent serves as an artificial substrate donating electrons and thereby becoming oxidized to a deep purple compound in the presence of the enzyme oxidase and free O2. Development of pink, then the desert, and finally dark purple coloration after rubbing the organism in the oxidase disc containing the reagent indicates a positive reaction. The positive attitude involves the conversion of colorless, reduced tetramethyl-p-phenylenediamine to oxidized form into deep purple color in the presence of Cytochrome C oxidase. No color change is indicative of the negative test result.

Gram staining is restricted almost exclusively to bacteria, and this is one of the limitay=tions of this procedure. Only a few other groups of organisms, for example, yeast are exhibit this reaction. Gram staining is quantitative variation, and gram positivity exists between different species. Sometimes gram staining technique may be faulty in that it is possible to report contrary if the gram stain is positive. This happens when the bacteria are old, dead, or damaged; therefore, their cell wall is not intact. False-positive can also occur if the decolorization step is omitted. Common errors committed during this procedure include the following, excessive heat during fixation, low concentration of crystal violet, excessive washing between the levels. Insufficient exposure of the iodine. Prolonger decolorization and excessive counterstaining. (9) Gram stain positive culture-negative specimens may also be the rest of the contamination of reagents and other supplies. Limitations of the catalase test include inconsistency inputting the correct and same amount

1. References

(1) Microbiology (2016) Catalase Principle test Pg 4 https://microbiologyinfo.com/catalase-test-principle-uses-procedure-result-interpretation-with-precautions/

(2) Taylor & Achanza (1972) Catalase test as an aid to the identification of Enterobacteriaceae applied microbiology https://aem.asm.org/content/24/1/58.short

(3) K. Reiner (2010) Catalase Test Protocol Microbial Library https://asm.org/getattachment/72a871fc-ba92-4128-a194-6f1bab5c3ab7/Catalase-Test-Protocol.pdf

(4) DT Petrico (1973) Capillary tube Catalase Test Pg 5 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC379865/

(5) PD Cotter, (2003) Microbiology and molecular biology Surviving the acid test responses of gram Pg 78-6

(6) G. Cerny(2016) European Journal of Applied Microbiology Studies on aminopeptidase test for the distribution of gram-positive bacteria Vol. 5

1. BM Johnson (2015) In vitro activity In vitro activity of tigecycline against 6792 Gram-negative and Gram-positive clinical isolates from the global Tigecycline Evaluation and Surveillance Trial Pg 6
2. Gregersen (1978) Rapid method for the Distinction of Gram-Negative from Gram-Positive European Journal of Applied Microbiology

(9) The Oxidase Test of Kovacs(1956) The Oxidase Test as A taxonomic Tool the journal of General Standard Operating Procedure Vol. 3