Wednesday, March 27, 2019

Aseptic Technique and the Transfer of Microorganisms

Objectives:


  1. To acquire the skill of aseptic technique in the field of Microbiology.
  2. To prevent contamination of cultures and media from microbes in the environment.
  3. To transfer cultures from one medium by inoculating another medium. This is called subculturing.
  4. To isolate a microorganism from a mixed culture to obtain a pure culture.
  5. To prevent lab microorganisms from being spread in the environment and/or infecting the investigator. 
     

Principle: 



Aseptic technique is a fundamental and important laboratory skill in the field of microbiology. Microbiologists use aseptic technique for a variety of procedures such as transferring cultures, inoculating media, isolation of pure cultures, and for performing microbiological tests. Proper aseptic technique prevents contamination of cultures from foreign bacteria inherent in the environment. For example, airborne microorganisms (including fungi), microbes picked up from the researcher’s body, the lab bench-top or other surfaces, microbes found in dust, as well as microbes found on unsterilized glassware and equipment, etc. may potentially contaminate cultures, thus interfering with the lab results. Using proper aseptic technique can greatly minimize or even eliminate the risk of contamination. In addition, aseptic technique is of utmost importance to maintain pure stock cultures while transferring cultures to new media. Aseptic technique is also essential for isolation of a single species of microorganism from a mixed culture to obtain a pure culture. Furthermore, proper aseptic technique prevents microbes used in the laboratory from accidentally being released into the environment and/ or infecting people working in the laboratory. This is especially relevant when pathogens are being handled.


 Significance of Flaming:


Flaming the loop :  Holding the loop in the flame of the Bunsen burner kills all contaminating organisms, thus sterilizing the loop.   The loop should glow red-hot for a few seconds. After flaming, make sure to slightly cool the loop before picking up organisms from the inoculum culture (the culture that is to be transferred.)  When transferring a culture from a plate, cool the loop by touching on the very edge of agar.  When transferring from a broth, the red-hot loop will make a sizzling noise as soon as   you insert it into the culture.  The loop will automatically cool once it makes contact with the broth culture, but wait a one or two seconds before removing the loopful of inoculum from the tube. (The hot loop may create aerosols when it touches the media containing microorganisms. It will cause some of the broth and bacteria to boil briefly, creating a bacteria-containing aerosol. This airborne bacteria have the chances of entering into the respiratory tract or into the body parts. If you hear a hissing sound when you place the heat sterilized loop into the broth culture indicates that the loop is not cooled sufficiently).

Flaming the Mouth of the Test Tube:  Passing the mouth of a tube through the flame of a Bunsen burner creates a convection current which forces air out of the tube.  This prevents airborne contaminants from entering the tube. The heat of the Bunsen burner also causes the air around your work area to rise, reducing the chance of airborne microorganisms contaminating your cultures.  

Agar Slants: Cultures are often transferred to agar slants, in addition to broth tubes and agar plates. An agar slant is a test tube containing agar, in which the solid agar forms a slant in the test tube.  When inoculating an agar slant, draw the loop containing the inoculum very lightly over the surface in a zigzag formation while being careful not to break the surface. A needle can be used instead of a loop to inoculate an agar slant by stabbing the needle containing the inoculum into the agar ( Fig 1).

Saturday, March 16, 2019

A List of Chemistry Laboratory Apparatus and Their Uses


A List of Basic Chemistry Apparatus

In most labs, you'll encounter the same basic apparatus. Here, you will find a picture and an explanation for how to use each piece of equipment. You will learn about:
  • Safety goggles and safety equipment
  • Beakers
  • Erlenmeyer flasks, AKA conical flasks
  • Florence flasks, AKA boiling flasks
  • Test tubes, tongs, and racks
  • Watch glasses
  • Crucibles
  • Funnels
  • Graduated cylinders
  • Volumetric flasks
  • Droppers
  • Pipettes
  • Burets
  • Ring stands, rings, and clamps
  • Tongs and forceps
  • Spatulas and scoopulas
  • Thermometers
  • Bunsen Burners
  • Balances

The Equipment You Will Encounter and Their Functions


Safety goggles and safety equipment
Safety goggles and safety equipment
The first and foremost rule of any laboratory is to be safe! This may seem obvious, but people often disregard safety protocols for one reason or another, putting themselves and those around them in danger. The best thing you can do is to make sure you follow all safety protocols at all times.
Safety goggles are required wear in all chemistry labs. Not wearing them puts you in danger of eye irritation and possibly blindness in the case of an accident. A small droplet of acid could splash out of the container at any time. Better safe than permanently blinded!
Latex gloves should be used when there is a possibility of corrosive chemicals spilling onto your hands.
A lab apron or coat can also prevent injury in case of spills or splashes.
Never wear open-toed shoes or sandals in a lab.

Beakers
Beakers
A beaker is a common container in most labs. It is used for mixing, stirring, and heating chemicals. Most beakers have spouts on their rims to aid in pouring. They also commonly have lips around their rims and markings to measure the volume they contain, although they are not a precise way to measure liquids. Beakers come in a wide range of sizes.
Because of the lip that runs around the rim, a lid for a beaker does not exist. However, a watch glass (discussed below) can be used to cover the opening to prevent contamination or splashing.

Erlenmeyer flasks, AKA conical flasks
Erlenmeyer flasks, AKA conical flasks
Also known as a conical flask, the Erlenmeyer flask was named after its inventor in 1861. It has a narrow neck and expands toward its base. This allows easy mixing and swirling of the flask without too much risk of spilling. The narrow opening also allows for the use of a rubber or glass stopper. It can easily be clamped to a ring stand (discussed below) as well as heated or shaken mechanically.
Once again, the marks on the side are meant primarily for estimation rather than precision.
An important safety tip here is to never heat this flask while it is capped. This could cause a pressure build-up that could result in explosion.

Florence flasks, AKA boiling flasks
Florence flasks, AKA boiling flasks
Also known as a boiling flask, the Florence flask has a round bottom and a long neck. It is used to hold liquids and can be easily swirled and heated. It can also easily be capped by rubber or glass stoppers.
Once again, safety dictates that this flask never be heated when capped. Pressure build-up and explosions can and do occur.

Test tubes being lifted with tongs from a rack
Test tubes being lifted with tongs from a rack
A test tube is a glass tube with one end open and the other end closed. The closed end is rounded. Test tubes are used to hold small samples. They are primarily used for qualitative assessment and comparison. A common place to see these is the biochemistry lab. When a large number of samples need to be tested and compared, test tubes are used to make this easier. They are also easily capped with a rubber or glass stopper.
They are generally held in a test tube rack specifically designed for the purpose. If the test tubes become unsafe to touch with bare hands (whether due to heat or another reason), test-tube tongs can be used to move them.
Never heat a capped test tube.

Watch glasses
Watch glasses
A watch glass is just a round piece of glass that is slightly concave/convex (think of a lens). It can hold a small amount of liquid or solid. They can be used for evaporation purposes and also can function as a lid for a beaker.

Crucibles
Crucibles
A crucible is a small clay cup made of a material that can withstand extreme temperatures. They are used for heating substances and come with lids.

Funnels
Funnels
A lab funnel is just like any other funnel except that it was designed to be used in a laboratory setting. They can be made of plastic or glass and can have either a short stem or a long stem, depending on what they are needed for. There are several sizes that can be chosen from based on the amount of liquid that needs to go through them quickly.

Graduated cylinders
Graduated cylinders
This is a primary measuring tool for the volume of a liquid. There are several markings up and down the length of the container with specific increments. Graduated cylinders come in many sizes. The smaller they are in diameter, the more specific the volume measurements will be.
When reading the volume from a graduated cylinder, you will notice that the liquid seems to have an indentation. The liquid around the edges will be higher than the liquid in the center, sloping down like the sides of a trampoline when someone is standing in the middle. This is called the meniscus. Line the lowest point of the meniscus up with the nearest marking, keeping the cylinder level to properly read the volume.

Volumetric flasks
Volumetric flasks
A volumetric flask is a round flask with a long neck and flat bottom. It is used to measure an exact volume of liquid. There is a small line on the neck that indicates how far to fill the bottle (use the bottom of the meniscus). They come with special caps that will not let anything in or out.
Remember that temperature affects volume; therefore avoid using liquids that will fluctuate in temperature (hot water that will cool, for example).

Droppers
Droppers
These are small glass tubes with narrow tips on one end and a rubber bulb on the other. They suck up liquid that can then be squeezed out in small drops. These can be used to add an indicator to a solution about to be titrated.

Pipettes
Pipettes
There are a large variety of pipettes designed to accomplish specific goals. However, they are all for measuring an exact volume of liquid and placing it into another container.

A buret. These are usually attached with a clamp to a ring stand, as shown in the picture below.
A buret. These are usually attached with a clamp to a ring stand, as shown in the picture below.
A buret is a glass tube that is open at the top and comes to a narrow pointed opening at the bottom. Right above the bottom opening is a stopcock that can be turned to control the amount of liquid being released. There are markings along the length of the tube that indicate the volume of liquid present.
A buret is used for extremely accurate addition of liquid. By adjusting the stopcock, the amount of liquid that is released can be slowed to a drop every few seconds. Burets are one of the most accurate tools in the lab.
Burets are set up by using a buret clamp in combination with a ring stand, discussed below.
To determine how much liquid is added, write down how much is initially in the buret. Then when you're finished adding, write down how much is left. Subtract the final amount from the initial amount and you have the volume of liquid added.
Again, remember to measure from the bottom of the meniscus!

Attaching a buret to a ring stand
Attaching a buret to a ring stand

Ring Stands, Rings, and Clamps


Ring stands with rings attached
Ring stands with rings attached
The ring stand is used to suspend burets, beakers, flasks, crucibles, etc. above other containers or, in some cases, a heat source (such as a Bunsen burner, discussed below).
Always make sure everything is clamped to the stand tightly. When clamping glass, be careful not to shatter the glass. Only tighten until snug.
When using a ring on the stand, there are usually other pieces necessary to accomplish the goal. Wire mesh is laid across the ring to distribute evenly heat and support the beaker. A clay triangle with an open center is used to suspend crucibles.
Make sure everything is balanced! Do not let the whole setup tip over.

Two tongs above and a pair of forceps below
Two tongs above and a pair of forceps below
Tongs and forceps are for grabbing things that should not be touched by hand. Some tongs are specially made to hold beakers, others to hold test tubes, and so on. There are also general tongs.
Forceps are used to grab small things like solid chemicals that are broken into chunks, so they can be safely handled and added to containers.

Three scoopulas on the left and a number of spatulas to the right.
Three scoopulas on the left and a number of spatulas to the right.
Spatulas and scoopulas are for scooping solid chemicals. They are typically used to scoop a chemical out of its original container onto a weigh boat so that it can be weighed on a balance.

Thermometers
Thermometers
A laboratory thermometer is used for measuring the temperature of liquids. It can be made of glass or it can be a thermocouple made of different metals.

An unlit bunsen burner connected to a gas source
An unlit bunsen burner connected to a gas source
A Bunsen burner is a mechanical apparatus that is connected to a flammable gas source. There is a knob to adjust the amount of gas flow and a rotating collar that controls airflow. These both must be adjusted to get an ideal flame for heating purposes. The burner is lit with a striker.
Utmost safety is required when using a Bunsen burner.
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Balances
Balances
A balance is used to weigh chemicals. The chemicals are always in some form of container and never placed directly on the balance. It is important not to move a balance because they have been calibrated for the exact position they are in. Some balances have plastic housing with small doors to keep air currents from affecting the measurement. Close these doors whenever the balance is in use.
To use a balance to determine the weight of a chemical, first put the empty container that the chemical will be in on the balance. Once you have a reading, press the "tare" or "zero" button on the balance. Remove the container from the balance and add the chemical (never add chemicals to a container while it is on the balance). Reweigh after adding the chemical to find the weight of only the chemical.
It is important to keep the balance clean.

Sunday, March 10, 2019

Differences Between Plasmodium falciparum and Plasmodium vivax

Malaria parasites are sporozoans and belongs to order Haemosporida. The genus Plasmodium has been subdivided into nine subgenera, of which three are found in mammals, four in birds and two in lizards. According to the classification, Plasmodium vivax, Plasmodium ovale and Plasmodium malariae belong to the subgenus Plasmodium, and Plasmodium falciparum belongs to the subgenera Laverania.
Differences Between Plasmodium falciparum and Plasmodium vivax
Differences Between Plasmodium falciparum and Plasmodium vivax with characters are given below:

Characteristics

P. faciparum

P. vivax

Duration of asexual phase in man

36-48 hrs
Usually 48 hrs
48 hrs

Duration of sporogony in mosquito

22-23 days at 20°C
10-12 days at 27°C
30 days at 17.5°C
10 days at 25-30°C

Duration of intrahepatic phase

5.5 days8 days

Duration of Schizogony

12 days14 days

Forms found in the smear

Rings and banana shaped gametocytesTrophozoites, schizonts and gametocytes

Level of usual maximum parasitemia

May exceed 200,000/µl, commonly 50,000/µlUp to 30,000/µl of blood

No. of merozoites released per infected hepatocyte

30,00010,000

Red cell preference

Younger cells (but can invade cells of all ages)Reticulocytes and red cells up to 2 weeks old

Parasitized Red cells

Not enlarge. Coarse stippling (Maurer’s clefts)Enlarged, pale. Fine stippling (Schuffner’s dots)

Pigment Color

Black and Dark BrownYellow or Golden Brown

Ability to cause relapses

NoYes

Ring Stage Trophozoite

Small rings (1/5 red cell diameter).
Often two granules.
Multiple rings common.
Large rings (1/3 to 1/2 diameter).
Single chromatin granule.
Two rings in one cell.

Pigment in developing trophozoites

Coarse, black, few clumpsFine, Light brown, scattered

Late Trophozoite

Compact
Medium Sized
Rarely amoeboid
Vacuole inconspicuous.
Pleomorphic
Large
Markedly amoeboid
Vacuole prominent.

Schizont

Small, compact,
Single pigmented mass
Seldom seen in the peripheral blood smear
Large, amoeboid,
Pigments coarse
Can be seen in the blood smear

Mature Schizont (segmenters)

8-16 (Usually 8-18).12-14 (Usually 12-18)

Microgametocytes

Kidney-shaped with blunt round ends.
Cytoplasm stains pale blue.
Nucleus large.
Chromatin diffuse.
Granules fine, scattered.
Spherical, compact.
Cytoplasm stains pale light blue.
Chromatin undivided.
Granules abundant.

Macrogametocytes

Crescentic.
Cytoplasm stains dark blue.
Nucleus compact.
Chromatin central.
Pigment more compact.
Spherical.
Cytoplasm stains dark blue.
Nucleus small.
Pigment diffuse ecoarse.

Disease

Malignant tertian malariaBenign tertian malaria

Differences Between Thick Blood Smear and Thin Blood Smear

Differences Between Thick Blood Smear and Thin Blood Smear

S.N.

Thick Blood Smear

Thin Blood Smear

1.

Thick blood smears are most useful for detecting the presence of parasites.Thin blood smears helps to discover which species of parasite is causing the infection.

2.

A thick blood smear is a drop of blood on a glass slide.A thin blood smear is a drop of blood that is spread across a large area of the slide.

3.

The blood films must be laked before or during staining to rupture all the RBC so that only WBC, platelets and parasites are visualized.The purpose is to allow malarial parasites to be seen within the RBC and to assess the size of the infected RBCs compared to uninfected RBCs

4.

Thick smears allow a more efficient detection of parasites (increased sensitivity 11 times than thin smear).Less sensitive than a thick film especially where there is a low parasitemia.

5.

It is not fixed in methanol.It is fixed in methanol.

6.

Thick smears are mainly used to detect infection and to estimate parasitemia.Thin smears allow the examiner to identify malaria species, quantify parasitemia, and recognize parasite forms like schizonts and gametocytes.

Benedict’s Test- Principle, Composition, Preparation, Procedure and Result Interpretation

Benedict’s Test is used to test for simple carbohydrates. The Benedict’s test identifies reducing sugars (monosaccharide’s and some disaccharides), which have free ketone or aldehyde functional groups. Benedict’s solution can be used to test for the presence of glucose in urine. 
Some sugars such as glucose are called reducing sugars because they are capable of transferring hydrogens (electrons) to other compounds, a process called reduction. When reducing sugars are mixed with Benedicts reagent and heated, a reduction reaction causes the Benedicts reagent to change color. The color varies from green to dark red (brick) or rusty-brown, depending on the amount of and type of sugar.
Benedict's Test
Benedict’s quantitative reagent contains potassium thiocyanate and is used to determine how much reducing sugar is present. This solution forms a copper thiocyanate precipitate which is white and can be used in a titration. The titration should be repeated with 1% glucose solution instead of the sample for calibration

Principle of Benedict’s Test

When Benedict’s solution and simple carbohydrates are heated, the solution changes to orange red/ brick red. This reaction is caused by the reducing property of simple carbohydrates. The copper (II) ions in the Benedict’s solution are reduced to Copper (I) ions, which causes the color change.
The red copper(I) oxide formed is insoluble in water and is precipitated out of solution. This accounts for the precipitate formed. As the concentration of reducing sugar increases, the nearer the final colour is to brick-red and the greater the precipitate formed. Sometimes a brick red solid, copper oxide, precipitates out of the solution and collects at the bottom of the test tube.
Sodium carbonate provides the alkaline conditions which are required for the redox reaction. Sodium citrate complexes with the copper (II) ions so that they do not deteriorate to copper(I) ions during storage.
Complex carbohydrates such as starches DO NOT react positive with the Benedict’s test unless they are broken down through heating or digestion (try chewing crackers and then doing the test). Table sugar (disaccharide) is a non-reducing sugar and does also not react with the iodine or with the Benedict Reagent. Sugar needs to be decomposed into its components glucose and fructose then the glucose test would be positive but the starch test would still be negative.

Composition and Preparation of Benedict’s Solution

Benedict’s solution is a deep-blue alkaline solution used to test for the presence of the aldehyde functional group, – CHO.
Anhydrous sodium carbonate = 100 gm
Sodium citrate – 173 gm
Copper(II) sulfate pentahydrate = 17.3 gm
One litre of Benedict’s solution can be prepared from 100 g of anhydrous sodium carbonate, 173 g of sodium citrate and 17.3 g of copper(II) sulfate pentahydrate.

Procedure of Benedict’s Test

  1. Approximately 1 ml of sample is placed into a clean test tube.
  2. 2 ml (10 drops) of Benedict’s reagent (CuSO4) is placed in the test tube.
  3. The solution is then heated in a boiling water bath for 3-5 minutes.
  4. Observe for color change in the solution of test tubes or precipitate formation.

Result Interpretation of Benedict’s Test

If the color upon boiling is changed into green, then there would be 0.1 to 0.5 percent sugar in solution.
If it changes color to yellow, then 0.5 to 1 percent sugar is present.
If it changes to orange, then it means that 1 to 1.5 percent sugar is present.
If color changes to red,then 1.5 to 2.0 percent sugar is present.
And if color changes to brick red,it means that more than 2 percent sugar is present in solution.
Result Interpretation of Benedict's Test
Positive Benedict’s Test: Formation of a reddish precipitate within three minutes. Reducing sugars present. Example: Glucose
Negative Benedict’s Test: No color change (Remains Blue). Reducing sugars absent. Example: Sucrose.

References

  1. National Institutes of Health, Testing for Lipids, Proteins and Carbohydrates- Benedict’s solution.
  2. Fayetteville State University- Biological Molecules: Carbohydrates, Lipids, Proteins.
  3. Harper College- Benedict’s Test.
  4. National Biochemicals Corp.- BENEDICT’S SOLUTION (MB4755).
  5. Science Olympiad- Use of Benedict’s Solution.
  6. Brilliant Biology Student 2015- Food Tests- Benedict’s Test for Reducing Sugars.
  7. BBC Bitesize- Chemistry- Carbohydrates.
  8. University of Manitoba- The Molecules of Life: Biochemistry- Carbohydrates.
  9. Northern Kentucky University- Benedict’s Reagent: A Test for Reducing Sugars.
  10. KNUST Open Educational Resources, Benedict’s Test – Qualitative Test in Carbohydrates.
  11. Mark Rothery’s Biology Web Site- Biochemical Tests.
  12. All Medical Stuff- Benedict’s test for reducing sugar.
  13. Hendrix College- Benedicts Test for Glucose.
  14. Info Please- Benedict’s solution.
  15. Mystrica- Benedict’s Test.
  16. Amrita Virtual Lab Collaborative Platform- Qualitative Analysis of Carbohydrates.
  17. Wikipedia.

Nutrient Agar: Composition, Preparation and Uses

Nutrient Agar is a general purpose, nutrient medium used for the cultivation of microbes supporting growth of a wide range of non-fastidious organisms. Nutrient agar is popular because it can grow a variety of types of bacteria and fungi, and contains many nutrients needed for the bacterial growth.
Composition of Nutrient Agar
  • 0.5% Peptone

It is an enzymatic digest of animal protein. Peptone is the principal source of organic nitrogen for the growing bacteria.
  • 0.3% beef extract/yeast extract

It is the water-soluble substances which aid in bacterial growth, such as vitamins, carbohydrates, organic nitrogen compounds and salts.
  • 1.5% agar

It is the solidifying agent.
  • 0.5% NaCl

The presence of sodium chloride in nutrient agar maintains a salt concentration in the medium that is similar to the cytoplasm of the microorganisms.
  • Distilled water

Water is essential for the growth of and reproduction of micro-organisms and also provides the medium through which various nutrients can be transported.
  • pH is adjusted to neutral (7.4) at 25 °C.

Preparation of Nutrient Agar
1. Suspend 28 g of nutrient agar powder in 1 litre of distilled water.
2. Heat this mixture while stirring to fully dissolve all components.
3. Autoclave the dissolved mixture at 121 degrees Celsius for 15 minutes.
4. Once the nutrient agar has been autoclaved, allow it to cool but not solidify.
5. Pour nutrient agar into each plate and leave plates on the sterile surface until the agar has solidified.
6. Replace the lid of each Petri dish and store the plates in a refrigerator.
Uses of Nutrients Agar
1. It is frequently used for isolation and purification of cultures.
2. It can also be used as a means for producing the bacterial lawns needed for antibiotic sensitivity tests.  In actuality, antibiotic sensitivity testing is typically performed on media specially formulated for that purpose.
Pictures
Four nutrient agar plates growing colonies of common Gram negative bacteria.
Four nutrient agar plates growing colonies of common Gram negative bacteria.
Source: Wikipedia