My Thesis: MATERIALS AND METHODS

CHAPTER 3

MATERIALS AND METHODS

 

3.1 Pharmaceutical Probiotic Products

 

Three brands of commercial pharmaceutical probiotic products of lyophilized form enclosed in capsules were purchased for this study. Description and composition labeled on these products are listed in Table 3.1. These products were purchased from the pharmacies around Klang valley before their expiration date. The products were stored at refrigerator 4°C until use.

 

Products	Form	Probiotics	Viability (CFU/capsule)
A	capsule	L .acidophilus	4x109
B	capsule	L. acidophilus, L. bulgaricus ,L. casei L .rhamnosus, B. bifidum, B. infantis, B. longum.	5x109
C	capsule	L. acidophilus, L. bulgaricus, L. casei, S. thermophilus, B. longum.	1x1010

 

Table 3.1: The pharmaceutical probiotic products used in this study.

 

 

3.2 Culture Media and Reagents

 

MRS agar (de Man-Rogosa Sharpe agar; Oxoid Ltd., Hamphire, England) was purchased from Oxoid ltd. Bile bovine, trypan blue and sodium propionate were purchased from Sigma Chemical Co. (Germany). Lithium chloride was purchased from AJAX Finechem Ltd. The phosphate buffer saline (PBS) was used as the buffer solution for serial dilution and was formulated from 0.144 g/L of KH₂PO₄, 9 g/L of NaCl and 0.795 g/L of Na₂HPO₄ (Fisher Scientific Inc.(US). Hydrochloric acid was purchased from Fisher Scientific Inc.(US).

 

 

3.3 Media preparation

 

MRS agar

MRS agar was prepared according to manufacturer instruction. An amount of 31g MRS (solid-powder) was added to 500mL distilled water in a Schott bottle and mixed thoroughly. Then, the mixture was autoclaved at 121°C for 15 minutes. MRS agar contains polysorbate, acetate, magnesium and manganese, which are known to act as special growth factors for lactobacilli, as well as a rich nutrient base. Then, MRS agar (liquid) was poured into sterilized Petri dishes. About 10 to15 mL of MRS agar (liquid) were sufficient for a single Petri dish. The agar dishes were allowed to solidify and stacked invert. MRS-agar (solid) plates were use directly or packed in plastic beg and stored in refrigerator.

 

 

MRS-bile agar

The formulation of MRS-bile was prepared according to Vinderola et al. (1999) where 0.75g (0.15%) of bile and 31g MRS (solid-powder) were added to 500mL distilled water in a Schott bottle. The mixture was mixed thoroughly and autoclaved at 121°C for 15 minutes. Next, sterilized MRS-bile agar (liquid) was poured into sterilized Petri dishes. About 10 to15 mL of MRS-bile (liquid) was sufficient for a single Petri dish. The MRS-bile agar (solid) plate was allowed to solidify and stacked in invert position (Vinderola et al., 1999; Vinderola et al., 2000).

 

MRS-LP agar

The formulation of MRS-bile was prepared according to Vinderola et al. (1999) where 0.2% of lithium chloride (solid-powder) and 0.3% of sodium propionate (solid-powder) were added to the MRS basal medium. 1g of lithium chloride and 1.5g of sodium propionate were added to 31g MRS (solid-powder) were added to 500mL distilled water in a Schott bottle (Vinderola et al., 1999; Vinderola et al., 2000). The mixture was mixed thoroughly and autoclaved at 121°C for 15 minutes. Next, sterilized MRS-LP agar (liquid) was poured into sterilized Petri dishes. About 10 to15 mL of MRS-LP (liquid) was sufficient for a single Petri dish. The MRS-LP agar plate was allowed to solidify and stacked in invert position.

 

 

3.4 Sterilization of Glassware and Aseptic Environment

 

The materials and apparatus were sterilized before use. Eppendorf tubes were placed in a beaker and covered with aluminum foil for autoclave. Universal bottles, micropipette tips, PBS and tubes rack were autoclaved at 121°C for 15 minutes.

 

The plating method was conducted in laminar flow. For aseptic environment, laminar flow was sterilized ultra violet (UV) light for 15 minutes. Subsequently, vortex, Bunsen burner, micropipette and all the glass ware were wiped with methylated spirit before use.

 

 

3.5 Viability of Pharmaceutical Probiotic Products

 

3.5.1 Plate Count Method

 

For total viable counts of each product, the probiotics powder was taken out from the capsule. Instantly, 0.1g of the homogenized powder was diluted with 9ml of sterilized PBS and vortex (Boeco, Germany) thoroughly. Subsequently, 0.1 ml of the aliquot was transferred to 0.9 ml of PBS in Eppendorf tube for serial dilution. The dilution was made for 10⁷-fold. Then, 0.1ml of the appropriate dilution was spread plated on the MRS agar, MRS-bile and MRS-LP, respectively (appendix 1). For each media, triplicate results were obtained.

Plates of MRS agar were incubated at 37°C under anaerobic and aerobic condition for 48 hours. Plates of MRS-bile agar were incubated at 37°C under aerobic for 48 hours. Plates of MRS-LP agar were incubated at 37°C under anaerobic condition for 48 hours. The anaerobic condition was achieved by use of an anaerobic jar (Merck Ltd., Darmstadt, Germany) and Anaerocult® A gas pack (Merck Ltd., Darmstadt, Germany). Anaerocult® A gas pack (Kieselguhr, iron powder, citron acid and sodium carbonate) bind to oxygen efficiently to create anaerobic condition.

 

 

3.5.2 Direct Count Method: Dye Exclusion Test

 

This staining method is based on "dye exclusion" cells where intact membranes do not take up the dye and are considered viable. Trypan blue staining with hemocytometer provides a method for distinguishing between viable and nonviable cells. The chemicals used were Trypan blue (0.4%) and PBS.

 

An amount of 1g of probiotics powder was added to 9ml of PBS in a universal bottle. Then, 0.1ml from the diluted sample was added to 0.9ml of PBS in Eppendorf tube for serial dilution. The serial dilution was performed until 10⁷-fold of dilution was obtained. For each dilution, 0.5ml of trypan blue and 0.3ml of PBS was added to 0.2ml of diluted sample. The mixture was allowed to stand for 5 to 15 minutes. One drop of the mixture was transferred with Pasteur pipette to the hemocytometer. The viable cells which are unstained and nonviable cells which are stained were observed under microscope (Nikon, Japan). The observation was recorded.

 

The similar test was repeated by centrifuged the probiotics with PBS suspension prior to serial dilution. The PBS was added to probiotics powder and were centrifuged at 13 000 rpm for 1 minutes. The sample was washed with PBS for three times. Then, the sample was serially diluted and stained with trypan blue. One drop of the sample was transferred to hemocytometer. The observation was recorded.

 

Calculation:

Cell viability (%) = total viable cells (unstained) X 100

total cells(stained and unstained)

 

 

 

3.6 Preparation of Low pH PBS

 

The pH of PBS was adjusted to pH 2, 3, 4 and 6 with 0.1 N acid hydrochloric (HCl) using Model 3501 pH meter (Lin et al., 2006; Shuhaimi et al., 1999). The acidified-PBS was autoclaved as 121°C for 15 minutes. The solution was allowed to cool to room temperature before the assay.

 

 

 

3.7 Acid Tolerance Test Simulated low pH

 

One gram homogenized probiotics powder was mixed with 9 ml of acidified-PBS of pH 2.0, 3, 4 and 6 respectively (Lin et al., 2006; Shuhaimi et al., 1999; Corcoran et al., 2005). For each pH of acidified-PBS, the mixture was incubated at 37°C for 0 hour, 1.5 hours and 3.0 hours. After incubation, viable bacterial counts were determined by plating serial dilutions (with PBS, pH 7.2) on MRS agar, MRS-bile and MRS-LP. The assay was performed in triplicate.

 

 

3.8 Morphology Identification

 

    The colonies were selected randomly for Gram staining. All colonies were confirmed as Gram positive with rod shape bacteria under microscope (1000X).

 

 

3.9 Statistical Analysis

 

The data were calculated with mean values and standard deviations in triplicate trials. Data generated from the experiments were analyzed for significance by the ANOVA-one way and Tukey's for multiple comparisons. Differences were considered significant at P<0.05.