LAB 3 : PREPARATION AND STERILIZATION OF CULTURE MEDIA

Laboratory's Information :

Laboratory's Number - LAB A203

Laboratory Title : PREPARATION AND STERILIZATION OF CULTURE MEDIA

Lecturer's Name : Associate Professor Dr. Liong Min Tze

Laboratory Assistant : Madam Najmah

Group Members : Charles Ng Wai Chun, Siti Hawa Binti Ramli, Nuramirah Binti Ramlan

Date of Laboratory : 13 October 2015

LAB 3 : PREPARATION AND STERILIZATION OF CULTURE MEDIA

Introduction

Microbes require nutrients to grow. These are supplied by either solid or liquid culture media. The standard solid medium is nutrient agar, a gelatinous substance derived from seaweed. The basic liquid medium is nutrient broth, typically a mix of water, meat extract peptone, and sodium chloride. The broth contains:

1.5 g/L “Lab-lemco” powder (a beef extract)

1.5 g/L yeast extract

5.0 g/L peptone (a nitrogen source)

5.0 g/L sodium chloride

15 g/L agar powder

The agar has the same composition, except that it contains 15 g/L agar. The final pH of media is 7.4.

An autoclave is a pressure chamber used to sterilize equipment and supplies by subjecting them to high pressure saturated steam at 121 °C (249°F) for around 15–20 minutes depending on the size of the load and the contents. It was invented by Charles Chamberland in 1879, although a precursor known as the steam digester was created by Denis Papin in 1679. The name comes from Greek auto-, ultimately meaning self, and Latin clavis meaning key—a self-locking device.

Sterilization autoclaves are widely used in microbiology, medicine, podiatry, tattooing, body piercing, veterinary science, mycology, funeral homes, dentistry, and prosthetics fabrication. They vary in size and function depending on the media to be sterilized. Typical loads include laboratory glassware, other equipment and waste, surgical instruments and medical waste.

A notable growing application of autoclaves is the pre-disposal treatment and sterilization of waste material, such as pathogenic hospital waste. Machines in this category largely operate under the same principles as conventional autoclaves in that they are able to neutralize potentially infectious agents by utilizing pressurized steam and superheated water. A new generation of waste converters is capable of achieving the same effect without a pressure vessel to sterilize culture media, rubber material, gowns, dressing, gloves, etc. It is particularly useful for materials which cannot withstand the higher temperature of a hot air oven.

Autoclaves are also widely used to cure composites and in the vulcanization of rubber. The high heat and pressure that autoclaves allow help to ensure that the best possible physical properties are repeatably attainable. The aerospace industry and sparmakers (for sailboats in particular) have autoclaves well over 50 feet (15 m) long, some over 10 feet (3.0 m) wide.

Objective

To prepare sterile nutrient agar for culturing microorganisms.

Materials and Reagents

1) Commercial Nutrient Agar

2) Brain Heart Infusion Broth (BHI)

3) Trypticase Soy Broth (TSAYE)

4) Peptone powder

5) Beef extract powder

6) Sodium chloride

7) Yeast extract

8) Electronic Weighing Balance

9) Measuring cylinder

10) Scott bottles

11) Distilled water

12) Beakers

13) Glass rod

Procedure & Results

1) Agar powder is weighted approximately in the beaker the it is dissolved with distilled water. The broth is mixed well.

2) The bottles are loosely recapped and set aside for sterilization.

3) All media is sterilized at 121°C for 15 minutes.

4) After autoclaving, the media is removed. The broth preparation is allowed to cool then the cap of each bottle is tight.

For step 1),

4 different types of culture media were prepared which are

I) Commercial nutrient agar which is made from 11.2g of commercial nutrient agar powder dissolved in 400ml of distilled water.

II) Trypticase Soy Agar (TSAYE) which is made from 4.0g of TSAYE agar powder dissolved in 100ml of distilled water.

III) Brain Heart Infusion Agar (BHI) which is made from 5.2g of BHI agar powder dissolved in 100ml of distilled water.

IV) Self-made nutrient agar powder which is made from these ingredients:

i.                0.6g of “Lab-lenco” powder

ii.               0.6g of yeast extract

iii.              2.0g of peptone

iv.              2.0g of Sodium chloride (NaCl)

v.               6.0g of agar powder

where all the ingredients above dissolved in 400ml of distilled water.

             

One of the ingredients in Self - made nutrient agar,
peptone, is weighed using an Electronic Weighing Balance

All the ingredients were well prepared for the self-made nutrient agar culture media..

All the culture media were prepared

Discussion

To prepare a good culture media with accurate nutrient content and with no contamination,there are several precaution steps that we need to take when conducting the experiment.

1) Balance (Electronic Weighing Balance)

• The precise and appropriate amount of broth powder and agar powder is weighed using electronic analytical balance which has the precision of one hundredth of a gram, ±0.01 or one ten-thousandth of a gram, ±0.0001 g.
• The surrounding of the pan and the pan of the balance must be clean without any debris or dusts. Place the receiver on the center of the pan of the balance and close the balance door. Then, press the appropriate tare key on the balance to set the signal from the strain gauge to zero so that the weight of the receiver is no longer indicated. With careful handling, add the powdered materials using a spatula until the desired amount is reached. Always handle the spatula filled with powdered materials with care to avoid spilling.
• If solids are spilled, remove the receiver and sweep out all of the spilled material from the balance using a brush.The spilled material must be properly disposed.

2) Autoclave (Autoclaving process)

• Before using the autoclave, check the drain screen at the bottom of the chamber.
• In order to have a high effectiveness of the autoclaving process, the autoclave must reach and maintain a temperature of 121-123 degree Celcius for at least 30 minutes. This is achieved by using saturated steam under at least 15 psi of pressure.
• All the debris must be removed and cleaned for efficient heat transfer as steam must flush out of the autoclave chamber. If the drain screen is blocked with debris, a layer of air may form at the bottom of the autoclave and prevent proper operation.   
• The water level should between range of low and high. If there are too low water level, water should be added in.
• The cap of the Scott bottles must not be too tight to prevent breakage off the Scott bottles.                      
• However, the cap of the Scott bottles must not be too loose to prevent the outflow of media from inside of the Scott bottles.   

• Autoclave doors must be firmly closed and locked into place before it starts running.                                                   
• Do not stack or store combustible material next to an autoclave (cardboard, plastic, volatile or flammable liquids).  
• Heat resistant gloves should be used when removing materials after sterilization to prevent any injure from the heat inside the autoclave.       
• Avoid touching the inner chamber surfaces after sterilization.
• To be effective the autoclave must reach and maintain a temperature of 121-123 degree Celcius for at least 30 minutes. This is achieved by using saturated steam under at least 15 psi of pressure.

.Scott bottles are ready to be sterilized in a autoclave.

3) Agar (Culture media)

• The ingredients and the uses of the agar have to be clearly known before we use it as the culture media.
• Commercial Nutrient agar is one of the most commonly used nutrient agar as culture media.
• Self - made Nutrient agar contains ingredients like "Lab-lemco" power (a beef extract), yeast extract, peptone, sodium chloride and agar powder is another agar used as culture media.
• Trypticase Soy agar (TSAYE) is a medium made with casein and soybean meal and is used as initial growth medium to observe bacterial morphology or increase bacterial growth for analysis or storage.
• Brain Heart Infusion (BHI) agar is a general purpose medium suitable for the cultivation of a wide variety of organism types, including bacteria, yeasts and moulds. The BHI agar derives its nutrients from the brain heart infusion, peptone and dextrose components. The peptones and infusion are sources of organic nitrogen, carbon, sulfur, vitamins and trace substances. Dextrose is the carbohydrate source that microorganisms utilize by fermentation action. The medium is buffered through the use of disodium phosphate.

Other precautions:

• Read the labels and instructions on the container before using them in preparing the culture media.                              
• Use distilled water to clean all the apparatuses that needed to be used in the experiment.                                                          
• Measuring cylinder is used to measure the volume of distilled water required accurately (Avoid using beaker to measure the volume of distilled water).
• Stir the mixture evenly to ensure that the nutrient powder dissolves completely.

Conclusion

       As a conclusion, from the experiment that we conducted, we have learned the correct techniques in preparing commercial and own recipe culture media based on the ingredient listed. Culture media must be stored at the specified temperature, under specified conditions such as pH and humidity . Exposure of sunlight to the culture media and their components has to be avoided . Moreover, the culture media must be sterilized before we can use it. One of the sterilizing methods is autoclaving which we were able to learn how to operate a autoclave machine with proper manners.

Reference

1) American Society for Microbiology (2014), Culture Media. Retrieved from http://www.microbeworld.org/careers/tools-of-the-trade/culture-equipment/culture-media

2) Wikipedia, the free encyclopedia (18 October 2015). Retrieved from https://en.wikipedia.org/wiki/Autoclave

3) Sagar Aryal (15 April 2015), Nutrient agar: Composition, Preparation and Uses. Retrieved from http://www.microbiologyinfo.com/nutrient-agar-composition-preparation-and-uses/

LAB 2 : MEASUREMENT AND COUNTING OF CELLS USING MICROSCOPE

Laboratory's Information :
Laboratory's Number - LAB A203
Laboratory Title : MEASUREMENT AND COUNTING OF CELLS USING MICROSCOPE
Lecturer's Name : Associate Professor Dr. Liong Min Tze
Laboratory Assistant : Madam Najmah
Group Members : Charles Ng Wai Chun, Siti Hawa Binti Ramli, Nuramirah Binti Ramlan
Date of Laboratory : 29 September 2015

LAB 2 : MEASUREMENT AND COUNTING OF CELLS USING MICROSCOPE

2.1 OCULAR MICROMETER 

Introduction

            An ocular micrometer is a glass disk that fits in a microscope eyepiece that has a ruled scale, which is used to measure the size of magnified objects under the microscope especially in measuring and comparing the size of prokaryotic and eukaryotic microorganisms. Suitable scales for their measurements should be somewhere in the microscope itself and the physical length of the marks on the scale depends on the degree of magnification. We must calibrate the ocular micrometer with the stage micrometer first before we want to calculate the size of a microorganism. We can use different types of magnifications to observe the samples we prepared but the calibration factors for the ocular micrometer is specific for each ocular objective combination because the objectives have different values of magnification. Therefore, each objective lens must be calibrated separately.

Steps in inserting a ocular micrometer into the eyepiece.

The ocular micrometer.

Objective

1) To measure and count cells using a microscope.

Materials and Reagents

1) Microscope fitted with an ocular micrometer.

2) Slide micrometer.

3) Stained preparation of yeast and bacteria.

Procedure

(refer to the laboratory manual)

Results

When the ocular micrometer is superimposed with stage micrometer under 40x magnification,

the calculation of one division\

50 division of stage scales superimpose with 20 divisions of ocular micrometer

1 division on stage scale = 50 x 0.01mm

                                        = 0.5mm

Therefore, one ocular division = 0.5mm / 20

                                                 = 0.025mm/25μm

When the ocular micrometer is superimposed with stage micrometer under 100x magnification,

the calculation of one division

10 divisions on stage scales superimposed with 10 divisions on ocular micrometer

1 division on stage scale = 10 x 0.01mm

                                        = 0.1mm

Therefore, one ocular division = 0.1mm / 10

                                                  = 0.01mm/10μm

For the size of one of the the yeast cells,
it covered half of an ocular division under 100x magnification,
so, its size will be

1/2 division x 0.01 mm = 0.005 mm 

                                      = 5μm

Discussion

1) Based on the experiment we conducted, we found that in order to measure the sizes of certain microorganisms, the ocular micrometer has to be calibrated with the stage scale to count the exactly length of each division in the eye piece.

2) The size of the scales of the ocular micrometer will not change as of what we can see, but the stage micrometer will change when different magnifications were applied. Therefore, there will be different values of one division under different magnifications.

3) Basically, ocular micrometer does not have units on them, so, to make use of the ocular micrometer , it has to be assigned or calibrated with the stage micrometer in order to get the the correct unit and scale

4) Then, the size of  yeast can be measured using the calibrated ocular micrometer.

Conclusion

            Ocular micrometer is a glass disk are oftenly use to measure the size of the cells easily. The exact size of a microorganism can only be determined by utilizing a calibrated ocular micrometer  It has a ruled scale and it fits in a microscope eyepiece. We can calculate the exact distance each ocular division measures on the microscopic field by determine how many units of the ocular micrometer superimpose a known distance on the stage micrometer.

Reference

1) OCULAR and STAGE MICROMETERS. Retrieved from http://fire.biol.wwu.edu/cmoyer/zztemp_fire/biol346_W06/labman_week2.pdf 

2) Wikipedia, the free encyclopedia, 8 June 2015. Retrieved from https://en.wikipedia.org/wiki/Ocular_micrometer

2.2 NEUBAUER CHAMBER

Introduction

            The neubauer chamber or hemocytometer is a device used to count cells. It is originally designed for the counting of bloods cells. The hemocytometer consists of a thick glass microscope slide with a rectangular indentation that creates a chamber. This chamber is engraved with a laser-etched grid of perpendicular lines. This device is carefully crafted so that the area bounded by the lines is known and the depth of the chamber is also known. It is therefore possible to count the number of cells or particles in a specific volume of fluid, and thereby calculate the concentration of cells in the fluid overall.

The hemocytometers.

Materials and Reagents

1) Serial dilutions of bacteria cultures.

2) Neubauer and coverslip.

3) 70% ethanol.

4) Sterile Pasteur Pipettes.

Procedure

(refer to the laboratory manual)

Results

Yeast cells on the hemocytometer.

10 results from randomly picked square boxes : 20, 19, 28, 27, 34, 29, 30, 33, 36, 26

The average number of 10 boxes = 282 / 10

                                                      = 28.2 cells

Volume of the square: 0.25 mm X 0.25 mm X 0.1 mm = 0.00625 mm³

0.00625mm³ / 1000 = 0.00000625cm³ = 0.00000625 mL 

There are 28.2 cells in 0.00000625 mL, 

thus concentration of the cells

 = 28.2 cells / 0.00000625 mL

 = 4512000 cells/mL

= 4.512 x 10⁶ cells/mL 

 Discussion

1) To count the number of cells using the Neubauer chamber, we must ensure that the coverslip is properly positioned on the surface of the counting chamber.
2) The yeast cells must be allowed to settle down before we observe and count the cells with the help of a microscope so that the yeast cells will not flow into the trough of the Neubauer chamber when we are in the process of counting the cells.
3) The randomly picked 10 square boxes from 25 square boxes has to be really random and avoid count the cells outside the square box.
4) If there are cells located between 2 square boxes, it should be counted once only and counting of the same cell twice has to be avoided so that the calculation be precise.

Conclusion

           Neubauer chamber is a thick crystal glass slide with two counting area separated by a H-shaped trough, by using the method of calculating the average of cells in a sample on the small boxes of chamber that we chosed randomly and we divided it to the volume of each box, we can calculate the concentration of the cells.

Reference

1) Celeromics. Cell Counting with Neubauder Chamber, Basic Hemocytometer Usage.  Retrieved from http://www.celeromics.com/en/resources/Technical%20Notes/cover-cell-glass.php

2) Rasayanika Biotechnology. Hemocytometer. Retrieved from http://www.rasayanika.com/product/lab-instruments/sigma_aldrich/hemocytometer/

3) Wikipedia, the free Encyclopedia. 2 February 2015. Retrieved from https://en.wikipedia.org/wiki/Hemocytometer

LAB 1 : PRINCILES AND USE OF MICROSCOPE

Laboratory's Information :
Laboratory's Number - LAB A203
Laboratory Title : PRINCIPLES AND USE OF MICROSCOPE
Lecturer's Name : Associate Professor Dr. Liong Min Tze
Laboratory Assistant : Madam Najmah
Group Members : Charles Ng Wai Chun, Siti Hawa Binti Ramli, Nuramirah Binti Ramlan
Date of Laboratory : 22 September 2015

LAB 1 : PRINCILES AND USE OF MICROSCOPE

1.1 SETTING UP AND USING THE MICROSCOPE

Introduction

            A microscope is an instrument used to see objects that are too small and to enlarge images of small objects that cannot be seen by naked eyes. The science of investigating small objects using such as instrument is called microscopy. “Micro” refers to tiny while “scope” refers to view. There are many types of microscope such as electron microscope, the ultramicroscope and the various types of scanning probe microscope. The most common is the optical microscope, which uses light to image the sample.

The Optical Microscope




Parts and Function of an Optiocal Microscope

Eyepiece	Contains the ocular lens, which provides a magnification power of 10x to 15x, usually. This is where you look through.
Nosepiece	Holds the objective lenses and can be rotated easily to change magnification.
Objective lenses	Usually, there are three or four objective lenses on a microscope, consisting of 4x, 10x, 40x and 100x magnification powers. In order to obtain the total magnification of an image, you need to multiply the eyepiece lens power by the objective lens power. So, if you couple a 10x eyepiece lens with a 40x objective lens, the total magnification is of 10 x 40 = 400 times.
Stage clips	Hold the slide in place.
Stage	It is a flat platform that supports the slide being analyzed.
Diaphragm	It controls the intensity and size of the cone light projected on the specimen. As a rule of thumb, the more transparent the specimen, less light is required.
Light Source	It projects light upwards through the diaphragm, slide and lenses.
Base	Supports the microscope.
Condenser lens	It helps to focus the light onto the sample analyzed. They are particularly helpful when coupled with the highest objective lens.
Arm	Supports the microscope when carried.
Coarse Adjustment Knob	When the knob is turned, the stage moves up or down, in order to coarse adjust the focus.
Fine Adjustment Knob	Used fine adjust the focus.

Magnification and Resolution

1) Basically, the microscope that we used in the laboratory has four different magnifications of objective lenses, which are, 4x objective lens, 10x objective lens, 40x objective lens and 100x objective lens which is also called oil-immersion lens.
2) The total magnification of the images that we will be seeing from the microscope is calculated by multiplying the objective lens power by the eyepiece lens power.
- The total magnification is calculated as follows :
i) 4x objective lens power X 10x eyepiece lens power = 40x magnification
ii) 10x objective lens power X 10x eyepiece lens power = 100x magnification
iii) 40x objective lens power X 10x eyepiece lens power = 400x magnification
iv) 100x objective lens power X 10x eyepiece lens power = 1000x magnification    
   
3) The ability to distinguish two very small and closely spaced object as separate entities known as resolution or resolving power. 
4) Closing of the diaphragm will cause the image contrast but decrease in its resolution while the opening of the diaphragm will decrease the contrast but increase its resolution. 
5) Ergo, the magnification and resolution are greatly related to each other and both of them are important in achieving a clear image that we wanted to see from a slide. 

         

                                           4x, 10x, 40x and 100x Objective lenses.

           10x Eyepiece lens. 

Objective

1) To know the ways to use and take care of a microscope correctly and properly.

2) To learn the importance of magnification and resolution of the microscope

Materials and Reagents

1) Microscope slide.

2) Cover slip.

Procedure

(refer to the laboratory manual)

Results

Lactobacillus under the 4x objective lens (40x magnification). 

    

 Lactobacillus under the 10x objective lens (100x magnification). 

                 

                                           

Lactobacillus under the 40x objective lens (400x magnification). 

    

  Lactobacillus under the 100x objective lens (1000x magnification). 

Discussion

1) When the Lactobacillus slide was observed under the 4x objective lens which is 40x magnification, we can only see the bacteria in a very small form which cannot be seen clearly.
2) When the Lactobacillus slide was observed under the 10x objective lens which is 100x magnification, the image formed is bigger than the previous objective lens and it is clearer as well.
3) When the Lactobacillus slide was observed under the 40x objective lens which is 400x magnification, we can roughly see the outline of the bacteria in a much more clearer form of image.
4) When the Lactobacillus slide was observed under the 100x objective lens (oil-immersion lens) which is 1000x magnification, the shape of the Lactobacillus can be seen and examined clearly. We can still roughly distinguish the bacteria even though it was overlapped. However, an electron microscope will be needed if we want to distinguish every bacteria in a slide.
5) Scientific classification of Lactobacillus :

    Scientific name : Lactobacillus
    Rank : Genus  
    Kingdom : Monera
    Division : Firmicutes
    Class : Bacilli
    Order : Lactobacillales
    Family : Lactobacillaceae

    Morphology of lactobacillus :
    - rod-shaped
    - convex
    - smooth
    - opaque without pigments

Conclusion

            Microorganisms are small living organism that needed to be magnified in order to be seen. Magnification helps us to see the objects seems bigger and meanwhile, resolution helps us to distinguish the two things from each others.

            Microscope is a very important instrument in aiding us seeing and examining the cells which cannot be seen with our naked eyes. And here are some precautions and steps that should be taken during the experiment:

1) Microscope is an expensive instrument which we need to handle it very carefully.               

2) When we are to examine a glass slide, always begin with the scanning (4x objective) or the low power objective (10x). Never begin with the high power objective (40X). It will be difficult to find what we are looking for under high power.

3) For better result, turning the coarse adjustment until the stage is as close to the objective as it will go.

4) Always clean the slides and microscope when finished the experiment. Be careful when handling glass slides and cover-slips.

Reference

1) Classification of Lactobacillus Acidophilus. Retrieved from 

http://cahoney-l-acidophilus.pbworks.com/w/page/6327452/Classification

2) Matthew K.Nelson (2011), Safe / Use of the microscope / Cladistics. Retrieved from http://www.natureboy.com/3454/labexercise1.pdf 

3) R. Bunemann & G. Wiliams (2008), Lactobacillus characteristics. Retrieved from http://www.coloss.org/beebook/I/gut-symbionts/4/4/2

4) Wikipedia, the free encyclopedia (3 October 2015), Microscope. Retrieved 

from https://en.wikipedia.org/wiki/Microscope

5) Wikipedia, the free encyclopedia (2 October 2015), Lactobacillus. Retrieved from https://en.wikipedia.org/wiki/Lactobacillus

1.2 EXAMINATION OF CELLS

Introduction

            Some if the specimens of bacteria cannot be identified directly under the microscope because of their extremely small in size. The use of low-power of high-power objective lenses are far not enough for the studying of the bacteria. In addition, staining or drying the cells for the studying of microorganisms will definitely distort them in terms of shapes and sizes, which eventually destroy them. Therefore, some specimens need to be suspended in a drop of liquid (such as immersion oil) for microscopic examination to fill the space between the cover slip and the slide which it allows light pass through it easily. Nonetheless, we can even determine whether the bacteria or cells are motile with this technique. And, this is known as “wet mount” technique. 

Objective

1) To provide an experience in the use of microscope.

2) To illustrate the diversity of cells and microorganisms.

Materials and Reagents

1) Culture

2) immersion oil

3) Lens tissue

4) A microscope slide containing stained microorganisms

5) Inoculating loop

6) Bunsen burner

7) Slide and coverslip

Procedure

(refer to the laboratory manual)

Results

Lactobacillus wet mount under the 100x objective lens (1000x magnification).

 Discussion

1) Under the 100x objective lens of 1000x magnification, we could see the shape of Lactobacillus which is rod-shaped. 

2) It can be clearly seen that the Lactobacillus was moving around the glass slide as well which proved its motility and was still alive.  

3) Lactobacillus is any of a group of rod-shaped, gram-positive, non-spore-forming bacteria of the family Lactobacillaceae.

4) Lactobacillus is a facultative anaerobe which they can produce energy through glycolysis and fermentation when oxygen is not present. 

5) Similar to other genera in the family, Lactobacillus is characterized by their ability to produce lactic acid as a by-product of glucose metabolism in its metabolism system.

6) Lactobacillus is generally non - motile and can survive in both aerobic and anaerobic environments. 

7) Lactobacillus is also a type of "friendly" bacteria that normally live in our digestive, urinary, and genital systems without causing disease. Lactobacillus is also used in some fermented foods like yogurt and in dietary supplements.

8) Commercial preparations of lactobacilli are used as probiotics to restore normal flora after the imbalance created by antibioric therapy.

Conclusion

            Based on the experiment we conducted, we found that using the wet mount technique with the aid of 100x objective lens or oil-immersion lens, we could see and examine the bacteria clearly including its size and shape. And from the experiment, we got to know that the Lactobacillus is a motile microorganism organism that have a rod-shape. This could be done because no staining or drying of specimens is needed and hence it maintain the shape and size of the bacteria without destroy it. Last but not least, to achieve the best condition of the specimens for the purpose of study, we must practise the aseptic technique during the preparation of the specimens in order to prevent contamination of the slides which may cause different results in our experiments. 

Reference

1) Oliver Kim (2008 - 2014), Making a wet mount microscope slide. Retrieved from http://www.microbehunter.com/making-a-wet-mount-microscope-slide/

2) David B. Frankhauser, Ph.D. (24 June 2005), PREPARATION OF WET MOUNT SLIDE. Retrieved from http://biology.clc.uc.edu/fankhauser/Labs/Microbiology/Wet_Mount/Wet_Mount.htm 

3) Wikipedia, the free encyclopedia (2 October 2015), Lactobacillus. Retrieved from https://en.wikipedia.org/wiki/Lactobacillus

4) WebMD (2005 - 2015), Find a vitamin or supplement LACTOBACILLUS. Retrieved from https://en.wikipedia.org/wiki/Lactobacillus

5) The Editors of Encyclopedia Britannica (2005), Lactobacillus bacteria. Retrieved from http://global.britannica.com/science/Lactobacillus