Microbiology

1 Identification of two unknown bacteria (culture-dependent growth)

MacConkey agar streak plate

Introduction

Based on the method of lactose fermentation the two kinds of bacteria that can be isolated or differentiated from one another which gram-negative bacteria and enteric bacteria and this method of differential culture medium and sections are known as the McConkeyy agar steak plate method. This agar is used for the isolation of gram-negative bacteria, also used in differentiating the lactose fermenting from the non-lactose fermenting bacteria, and lastly, it is highly used in knowing the isolation of coliforms and intestinal water pathogens and biological specimens (Ibrahim, & Hameed, 2015).

Method

Step 1 take one thousand ml of purified or distilled water and suspend the medium in it approx. 50 gm
Step 2 to dissolve the  added medium completely in it heat it so that it can boil and which will result in proper mixing of the substance
Step 3 use the autoclave to sterilize at around 15 lbs of pressure and also maintain 121 degrees Celsius for up to 15 minutes
Step 4 after all that it is time to let it cool down and that is up to between 45 to 50 degrees Celsius.
Step 5 and just before pouring it on the sterile Petri plates mix it well so that the result that is shown would be more accurate.

 

Result

Lactose fermenter does pink and turns red on the agar and those which are non fermenters do not show any sort of the color change in them based on this color-changing process it becomes easy to identify e coli from proteus mirabilis

Gram stain of bacteria from an isolated colony

Introduction

Gram stain is a process by which the positive and negative bacterias can be distinguished and identified it is a colored purple and on coming in contact with the bacteria colony it changes its color it doesn’t change the color or when it is purple even after coming in contact that indicates that the bacteria that is been in the sample are of gram-positive and those who change the color termed as the gram-negative bacteria. It is to differentiate the bacteria in the two varieties of the cell (Yang, et al., 2019).

Method

Step 1 take a clean slide of glass and put a few drops of sterile saline on it.

Step 2 uses a single bacterial colony. And dip the colony of bacteria in the saline and now make a thin film of it while spreading it properly

Step 3 let it dry

Step 4 fix the bacterial colony with the help of heat on the slide with the help of Bunsen flame and do it two to three times for a better result.

Step5 is the most important method as it includes the straining process use the right gloves and you could use any of the following written here for the purpose and time does change with different reagents for example reagent could be crystal violet, grams iodine. Etc.

Step 6 at the end of the test analyzes the film under the oil immersion lens from a microscope.

Step 7 based on the color that it forms results going to be accordingly to what you observe in it.

Result

After observing the color it produces when the test is over will tell the answer if the color changes rather than purple then it will consider gram-negative bacteria.

2 Identification of six unknown Enterobacteriaceae (biochemical and molecular)

Analysis of API 20E

Introduction

API 20E is the plastic strip that contains only 20 chambers for test and this all is in small size it also contains dehydrated media along with the chemically defined compositions for each one of the chamber. Usually, they are being used to detect the activity of the enzyme, and if talk more accurately used in related to the fermentation of carbohydrates of the catabolism of proteins or even for amino acids by the inoculated organism.

It is used for the identification of gram-positive and negative bacteria and also yeast, it has been observed based on the different tests that it is capable of delivering accurate identification based on extensive databases. The full form of API is an analytical profile index it is a panel for the identification of biochemicals (Gruzdys, V., et al 2020).

Method

1. API 20E identification strips were previously inoculated with 6 different isolates of bacteria.
2. 16S rRNA sequence data.

Procedure

1 to confirm the culture and to test, a quick oxidase test for cytochrome c oxidase has been performed.

2 we picked one of the slides among the 6 different present slides and made a suspension of distilled water which was sterilized too.

3 after that we have taken the API20E strip and then put the reagent into the 20 separate compartments which are present in the strip after that we used the Pasteur pipette, and filled it up with the suspension of bacteria.

4 Abd than after it we put the oil for sterile purposes into the ADH, LDC, ODC, H2S, and URE blocks.

5 we then added some of the water drops into the tray and added the API test strip and then just closed the tray.

6 after that we marked the tray with the number for the identification purpose so that we can remember which was it and don’t mix things up for most of the different things we put dates and initials to for more accuracy.

7 after all of that we incubate the tray at a constant temperature of 37 degrees Celsius about in between 18 to 24 hours and after that, they can provide the right desired result and are ready to observe (Dilnessa, et al., 2016).

Result

A – klebsiella pneumoniae

B – pseudomonas flurescens

C – Escherichia coli

D – Enterobacter colacae

E – proteus mirabilis

F – Serratia marcesens

For some of the cells in the tray, the color change will be the indicator and it is the first visible indicator just after the result. But before making any sort of interpretation some of the reagents has to be put into it. The reagent that will be used are TDA, and IND VP, this will require in very minor quantities.

After that mark the API reading scale which is a color chart based on positive and negative on the lid of the tray. And after that based on the result, the seven-digit score will be allocated after adding the value which will be shown in all three individuals and it will provide the 7-digit number.

ISOLATE

A

B

C

D

E

F

COLOUR

RESULT

VALUE

COLOUR

RESULT

VALUE

COLOUR

RESULT

VALUE

COLOUR

RESULT

VALUE

COLOUR

RESULT

VALUE

COLOUR

RESULT

VALUE

ONPG

W

N

0

Y

P

1

0

G

P

1

Y

P

1

V

P

1

ADH

P

P

2

0

0

P

P

2

LDC

Y

N

0

O

P

4

0

R

P

4

O

P

4

ADC

Y

N

0

0

R

P

1

R

P

1

P

P

1

CIT

B

P

2

0

BL

P

2

B

P

2

B

P

2

B

P

2

H2S

PW

N

4

0

BL

P

4

URE

Y

N

0

0

P

P

1

TDA

W

N

2

0

BL

P

2

BR

P

2

IND

LP

N

4

Y

P

4

0

VP

B

N

1

Y

P

0

LP

P

LP

P

1

GEL

Y

P

2

BL

P

2

B

P

2

GLU

G

P

4

Y

P

4

Y

P

4

Y

P

4

Y

P

4

Y

P

4

MAN

G

P

1

Y

P

1

Y

P

1

Y

P

1

Y

P

1

INFO

G

P

2

Y

P

2

Y

P

2

SOR

G

P

4

Y

P

4

YG

P

4

Y

P

4

Y

P

4

THE

G

P

1

G

P

1

Y

P

1

SAC

Y

P

2

Y

P

2

Y

P

2

Y

P

2

MEL

B

P

4

Y

P

4

Y

P

4

Y

P

4

AMY

Y

N

1

Y

P

2

Y

P

1

Y

P

1

Y

P

1

ARA

Y

P

2

Y

P

2

Y

P

2

Y

p

2

OX

NUMERICAL PROFILE

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Analysis of 16s rRNA

Introduction

In the name 16s rRNA, 16s stands for ribosomal ribonucleic acid, and in this S is for the Svedberg it is a unit for measurement. And the most important part of the small subunit of prokaryotic ribosomes is rRNA also for the mitochondria and also for the chloroplasts. Generally many get confused with the protein-coding gene is 16s RNA but it, not the ribosome is a must for a binding site which makes it able to produce the proteins (Abellan-Schneyder, et al., 2021).

In today’s time, rDNA sequence analysis or 16S rRNA is becoming the major tool of choice in terms of it helps in knowing the relation between bacteria, and it is used widely for identification. All the bacteria which are present out there is having 16s rRNA gene in them and are not the same but the somewhat similar form is present in all cells which also include the eukaryotic cell.

16s ribosomal databases are EzBioCloud, ribosomal database project, SILVA, and Green Genes.

Ezbiocloud database also known as ExTaxon has a total hierarchical taxonomic system and it has more than 60 k bacteria and archaea species variety and it is having valid data of bacteria of about 15 k which has been had published names.

A ribosomal data project is a kind of program which has data on bacteria along with the related services and programs regarding them. This data can be available by any e-mail server. And it is also used as a bioinformatic tool development because of the huge size it has that is for creating curated databases.

Result

The reason why it is highly conserved is that the 16s rRNA genes have been found in every prokaryotic cell, and all bacterias have it because it is not possible for an organism to translate mRNA without having 16s rRNA and it is a part of small subunit in ribosome which means all bacteria have it. The genes are essential that’s why. And very highly conserved (Church, et al., 2020).

3 Counting CFUs in Yakult and comparison to published data

Introduction

It is an energy drink or we can say it is a milk item from japan and it is sold by Yakult Honshu which is situated in Tokyo. This beverage is fermented with the help of a bacteria strain of lactobacillus casei Shirota.

CFU stands for the colony forming unit and it has been seen that Yakult has almost 20 billon colony forming units of lacticasebibacillus paracasei Shirota per 65ml. and the current experiment is to find the number of cfu present in the yakult and than comparing it with the data that is present already (Berger, et al., 2021).

Method

Step 1 bottom of the Petri plates must be labeled and  label four types ofsaline

SStep2 there are two techniques for this process in this one aseptic technique is being used the first dilution is made by transferring the e coli sample to a sterile saline blank.

Step 3 uncaps it immediately after the shaking quickly uncaps it and then transfers it aseptically to the second saline blank. It will show  the dilute the original sample

Step 4 very fast shake the dilution or shake it vigorously and just after that transfer it to the third blank. It will show the dilution of the original sample

Step 5 does the same process again shake it well and after that transfer, it from the first plate to the next one do the same for the other two dilutions.

Step 6 after that it is time for revoking one agar and adding it to the tube from the 50 degrees Celsius water bath. And then remove the cover from the first one and then add agar to it, you will observe that they are missing well or instantly let it rest. Repeat the process for all other plates which are available.

Step 7after the agar has been hard after pouring it in letting it cool its time for their incubation at 25 degrees Celsius for 2 days or one day with a slightly higher temperature.

Step 8 after the test is done it’s time for the calculation per milliliter and based on the result it became easy to analyze the actual data whether it’s nearly close to it or not from what it claims in official data.

Result

The data which has been come after the test is around approx. same from what the actual data has been provided and the test was a success which means.

Biochemistry

Carbohydrates trinders assay

Introduction

This reaction is the test or reaction between hydrogen peroxide and phenol and minoantipyrine which then forms quinone, and in the presence of peroxidase it gets catalyzed and the example of a catalyst can be horseradish peroxidase.

The activity test for glucose Trinder glucose activity test is used in medicine to check the presence of glucose or its oxidase form. The test includes the use of Trinder’s reagent, which is named after P. Trinder was in a biochemistry department of Sunderland in the royal infirmary (Genzen, et al., 2016).

In the above-mentioned reaction between hydrogen peroxidase and phenol in the quinone that formed is red-violet, and this is important to look at the color that has been coming out after the result of the reaction because the intensity of the color which is present in the result will tell about the amount of glucose content present in it.

Method

Carbohydrates trinders assay

Step 1 reagents aliquot in test tube made up of glass

Step 2 at 37 degrees Celsius incubate it for about 20 min

Step 3 by using the no glucose sample do a blank spectrophotometer at 515nm and then press 0.

Step 4 while not pressing E analyze the absorbance of each sample

Step 5 records the curves which are coming out as a result of the experiment and only uses the standard curve for knowing the amount of concentration of the unknown glucose

Result

As a result of the experiment, it helps in knowing the glucose level present in it. Clear water or solution of carbohydrates to be checked in a test tube and phenol with sulfuric acid is added then after. The solution turns yellow-orange as a result of the collision or contact between the phenol and carbohydrates.

Separation of lipids thin layer chromatography

Introduction

Thin layer chromatography also known as TLC is a very common and easy technique which makes it a very versatile technique for the separation of lipid classes. The correct resolution, and doesn’t require costly instruments (Hölzl, & Dörmann, 2021).

Method

Step 1 fix the TLC plate with a hotplate.

Step 2 uses a pencil to mark and mark the actual position.

Step 3 spot the changes in the lane when increasing 5ul and also allow it to dry

Step 4 do it multiple times

Step 5 now analyses the lipid extract in their lane while increasing the 5ul.

Step 6 put it inside the active TLC chamber, and closely tighten the lid.

Step 7 at the end of the test mark the solvent using a pencil and allow it to dry.

Step 8 now put at the iodine chamber

Step 9 at the end of the process remove and lightly outline the spot.

Result

The method was for the separation of the lipid and at the end of the result, it became very clear that TLC can be used to know the different compounds present in the mixture and also the identity and purity of the compound too.

References

Abellan-Schneyder, I., Matchado, M. S., Reitmeier, S., Sommer, A., Sewald, Z., Baumbach, J., … & Neuhaus, K. (2021). Primer, pipelines, parameters: issues in 16S rRNA gene sequencing. msphere, 6(1), e01202-20.

Berger, D. R., Centeno, C. J., Kisiday, J. D., McIlwraith, C. W., & Steinmetz, N. J. (2021). Colony Forming Potential and Protein Composition of Commercial Umbilical Cord Allograft Products in Comparison With Autologous Orthobiologics. The American Journal of Sports Medicine, 49(12), 3404-3413.

Church, D. L., Cerutti, L., Gürtler, A., Griener, T., Zelazny, A., & Emler, S. (2020). Performance and application of 16S rRNA gene cycle sequencing for routine identification of bacteria in the clinical microbiology laboratory. Clinical Microbiology Reviews, 33(4), e00053-19.

Dilnessa, T., Mengistu, G. D. G., & Bitew, A. (2016). Journal of Medical Microbiology & Diagnosis.

Genzen, J. R., Hunsaker, J. J., Nelson, L. S., Faine, B. A., & Krasowski, M. D. (2016). N-acetylcysteine interference of Trinder-based assays. Clinical biochemistry, 49(1-2), 100-104.

Gruzdys, V., Signorelli, H., & Johnson-Davis, K. L. (2020). NicAlert™ test strip performance comparison with LC-MS/MS and immunoassay methods for nicotine and cotinine. Archives of Clinical Toxicology, 2(2), 19-24.

Hölzl, G., & Dörmann, P. (2021). Thin-Layer Chromatography. In Plant Lipids (pp. 29-41). Humana, New York, NY.

Ibrahim, I. A. J., & Hameed, T. A. K. (2015). Isolation, characterization, and antimicrobial resistance patterns of lactose-fermenter Enterobacteriaceae isolates from clinical and environmental samples. Open journal of medical microbiology, 5(04), 169-176.

Temitope, O. O., Fasusi, A. O., Ogunmodede, A. F., & MI, E. (2016). ANTIBACTERIAL SENSITIVITY PROFILE, ISOLATION, AND IDENTIFICATION OF BACTERIA USING API 20E KITS FROM HOSPITAL SOLID WASTE DUMP SITES OF IKARE-AKOKO, STATE SPECIALIST HOSPITAL, ONDO STATE, NIGERIA.

Yang, J., Gao, G., Zhang, X., Ma, Y. H., Chen, X., & Wu, F. G. (2019). One-step synthesis of carbon dots with bacterial contact-enhanced fluorescence emission: fast Gram-type identification and selective Gram-positive bacterial inactivation. Carbon, 146, 827-839.