**4. Common media used in isolation and detection of bifidobacteria**

*Bifidobacterium* in Human GI Tract:

Screening, Isolation, Survival and Growth Kinetics in Simulated Gastrointestinal Conditions 295

group: 1 = non selective medium, 2 = medium without antibiotics but with elective carbohydrate,

3 = medium with antibiotics, 4 = medium with propionate, 5 = medium with elective substance and/or low pH **Table 3.** Media used for the detection of bifidobacteria from faeces (Source: Hartemink, 1999).

thoroughly examined and tested with different baby faeces.

between these 3 media is exceedingly little, actually less than one log unit.

Combinations and media belonging to more than one group are also used. From the large number of media used, it can be concluded that there is no standard medium for the detection of bifidobacteria. *Bifidobacterium* spp. in the GI tract of humans are normally present in an adequate amounts and estimated to be between 109 and 1010 colony forming units (CFU) per gram wet weight or around 3% of total microbiota (Jia *et al.*, 2010). However, the selectivity of independent media for the quantification of bifidobacteria is

The experimental results of 3 media (PROP, RB and NPLN) tested on bifidobacteria show a wide variation in counts for the different samples (see Figure 6). Absolute counts are highest for the faecal samples on NPLN, followed by RB in 8 of 9 samples. PROP showed the lowest counts. However, as it can be observed from the same Figure 6, the principal difference

**Medium Group** ٭ Acetylglucosamine - Lactose (AL) agar 2 Bifidobacterium selective (BS) agar 3, 5 Bifidobacterium selective medium (BBM) agar 2, 3 Bifidus Blood agar 5 Bifidobacterium Iodoacetate Medium (BIM-25) agar 3 China Blue agar 5 Liver Cysteine Lactose (LCL) agar 2 Rogosa agar 1 Modified Rogosa agar 3, 5 MPN-agar 2, 3 MRS 1 MRS agar with LiCl and antibiotics (MRS-NN) 3, 5 Neomycin Paromomycin Lithium Nalidixic acid (NPLN) agar 3, 4 Propionate agar or Beerens agar 4 Raffinose-Bifidobacterium (RB) agar 2, 4 Reinforced Clostridial agar with Cephalothin and blood (RCB) 3 Tomato Casein Peptone Yeast agar (TCPY) 5 Tomato Casein Peptone Yeast agar (TPCY) with azide 5 Tomato Casein Peptone Yeast agar (TPCY with sorbic acid 5 Tomato Casein Peptone Yeast agar (TPCY with antibiotics 3 Transgalactosyloligosaccharide (TOS - agar) 2 TTC-agar 5 x-Gal medium 5 YN-6 agar 2, 3, 5

Legends on Table 3: ٭


Many different media for bifidobacteria are outlined in Table 2.

Legends on Table 2: ٭iac = iodoacetic acid, kan = kanamycin, LiCl = lithiumchloride, nal = nalidixic acid, neo = neomycin, paro = paromomycin, prop = propionate, TOS = transgalactosyl oligosaccharides, TTC = 2,3,5-triphenyltetrazoliumchloride

**Table 2.** Popular media used for the enumeration of bifidobacteria from faeces, dairy- and pharmaceutical products, (Adapted from prebiotic effect on non-digestible oligo- and polysaccharides by Hartemink, 1999).

Media used for the detection of bifidobacteria can be classified in 5 different groups. These are non-selective medium (such as MRS and Rogosa), medium without antibiotics but with elective carbohydrate, medium with antibiotics, medium with propionate, and medium with elective substance and/or low pH (Table 3).


Bifidobacterium Iodoacetate Medium (BIM-25

Neomycin Paromomycin Lithium Nalidixic acid

tetrazoliumchloride

by Hartemink, 1999).

with elective substance and/or low pH (Table 3).

Many different media for bifidobacteria are outlined in Table 2.

**4. Common media used in isolation and detection of bifidobacteria** 

**Medium Selectivity based on**٭ **Used for**  Acetylglucosamine-Lactose (AL) agar lactose, acetylglucosamine faeces AMC-agar nal, polymyxin B, kan, iac, TTC, LiCl,

Bifidobacterium selective (BS) agar LiCl, neo, paro, prop faeces Bifidobacterium selective medium (BBM-agar) nal, rifampicine, raffinose faeces Bifidus Blood Agar aniline blue, blood faeces Bif-medium human whey, nal, paro, aztreonam,

agar) kan, nal, iac, neo, polymyxin B sewage BS-agar LiCl, neo, paro, prop faeces China Blue (CB) agar specific impact of china blue faeces GL-agar galactose, LiCl dairy products Liver Cysteine Lactose (LCL) agar lactose, liver infusion faeces LP agar lactose, LiCl, prop dairy products Modified Rogosa agar neo, paro, prop, LiCl dairy products MPN-agar lactose, nal faeces MRS-LP-agar prop, LiCl dairy products

Propionate or Beerens agar propionic acid, pH 5.0 faeces

**Table 2.** Popular media used for the enumeration of bifidobacteria from faeces, dairy- and

pharmaceutical products, (Adapted from prebiotic effect on non-digestible oligo- and polysaccharides

Media used for the detection of bifidobacteria can be classified in 5 different groups. These are non-selective medium (such as MRS and Rogosa), medium without antibiotics but with elective carbohydrate, medium with antibiotics, medium with propionate, and medium

(NPLN) agar LiCl, nal, neo, paro, prop faeces, dairy products

Raffinose-Bifidobacterium (RB) Agar raffinose, LiCl, propionate faeces, dairy products RCM (modified) low pH dairy products RCM + stain Loeffler's methylene blue stain dairy products Rogosa agar low pH faeces, dairy products Rogosa (modified) neo, paro, prop, LiCl dairy products Rogosa-N low pH, nal faeces Tomato Casein Peptone Yeast Agar (TCPY) tomato juice faeces

Transgalactosyloligosaccharide (TOS-Agar) TOS faeces, dairy products TOS-Agar (modified) TOS, nal, neo, paro dairy products TPYd-agar dicloxacillin dairy products TTC-agar TTC faecal contamination VF-agar (modified) LiCl, prop, neo, sodium lauryl sulfate dairy products YN-6 agar lactose, nal, neo, bromocresol green faeces, sewage Legends on Table 2: ٭iac = iodoacetic acid, kan = kanamycin, LiCl = lithiumchloride, nal = nalidixic acid, neo = neomycin, paro = paromomycin, prop = propionate, TOS = transgalactosyl oligosaccharides, TTC = 2,3,5-triphenyl-

prop *B. longum* 

netilmycin dairy products

Legends on Table 3: ٭ group: 1 = non selective medium, 2 = medium without antibiotics but with elective carbohydrate, 3 = medium with antibiotics, 4 = medium with propionate, 5 = medium with elective substance and/or low pH

**Table 3.** Media used for the detection of bifidobacteria from faeces (Source: Hartemink, 1999).

Combinations and media belonging to more than one group are also used. From the large number of media used, it can be concluded that there is no standard medium for the detection of bifidobacteria. *Bifidobacterium* spp. in the GI tract of humans are normally present in an adequate amounts and estimated to be between 109 and 1010 colony forming units (CFU) per gram wet weight or around 3% of total microbiota (Jia *et al.*, 2010). However, the selectivity of independent media for the quantification of bifidobacteria is thoroughly examined and tested with different baby faeces.

The experimental results of 3 media (PROP, RB and NPLN) tested on bifidobacteria show a wide variation in counts for the different samples (see Figure 6). Absolute counts are highest for the faecal samples on NPLN, followed by RB in 8 of 9 samples. PROP showed the lowest counts. However, as it can be observed from the same Figure 6, the principal difference between these 3 media is exceedingly little, actually less than one log unit.

*Bifidobacterium* in Human GI Tract:

Screening, Isolation, Survival and Growth Kinetics in Simulated Gastrointestinal Conditions 297

Most false positive colonies are reported to be different cocci (mono-, diplo- or streptococci), spore-forming rods and short rods. No yeast is observed on any of the media tested. Based on the actual counts, selectivity can only be determined for RB, as the colonies of bifidobacteria and non-bifidobacteria cannot be determined for the other media and not all colonies are tested for their morphology. Selectivity as percentages of non-typical colonies

Colonies of different shapes can be tested microscopically. Bacterial morphology is determined, and typical and non-typical morphology is also determined. Typical morphology of bifidobacteria is branched or bifid-shaped rods. For the determination of bifidobacteria, none of the 3 media tested was decidedly selective. In this study, the occurrence of false positive or false negative colonies was determined. The lowest incidence of potential false positive colonies was observed on NPLN, but in all 3 media, the number of non-bifidobacteria capable of growing on the selective media was remarkably high. When many different species are capable of growing on the medium, an increase of one of these species may result in serious mistakes in calculating bifidobacteria. NPLN and RB gave slight higher counts than PROP. The incidence of false positive, based on morphologies on RB was comparable with that on the PROP and slightly higher than that on NPLN. The incidence of competitive flora was relatively low (less than 10% of the total colonies on the

plates), as bifidobacteria are one of the main groups of intestinal bacteria in humans.

PROP medium has been described as the best medium for the determination of bifidobacteria by Silvi *et al*., (1996), but they also concluded that the total bifidobacterial counts were significantly lower on PROP than on the other media tested. Similarly, Favier *et al*., (1997) concluded that PROP underestimated bifidobacteria in some of their samples. Both studies used human faeces as the test substrate. Several other studies, in which PROP agar is used, also show significantly lower bifidobacterial counts than most other studies (Favier *et al*., 1997).

NPLN, which has been described as the medium of choice to choose bifidobacteria in dairy products, showed many cocci. This was in accordance with results observed by Silvi *et al*., (1996). In the same study, BIM-25 was tested, and this medium was found to be non-specific. All these 3 media performed reasonably well for human faeces and bifidobacteria can reliably be counted. The typical colonies morphological trait and the basic cellularmorphology of bifidobacteria were demonstrated well by RB media, with reference to NPNL and PROP medium. The RB medium presented strains with double thickness

diameter and more bifurcated cellular morphology under phase contrast microscopy.

**5. Experimental procedures for the enumeration of bifidobacteria and** 

LAB or bifidobacteria strains can be selected or isolated from commercial or alleged "own isolates" strains, from freeze-dried cultures which are resuscitated to stationary phase in MRS broth at a ratio of 2% of the volume of the fresh broth. Decimal dilutions are put onto Raffinose–Bifidobacterium (RB) agar plates whose pH had to be adjusted to 6.8 – 7.0 with 2

**determining microbial inactivation by low acidic pH or bile salts** 

ranges from around 5 – 7%.

**Figure 6.** Counts (log CFU/gm wet weight) on PROP, NPLN and RB media in babies' faeces.

Selectivity is also determined by microscopic observations of all different colony morphologies on all countable (between 10 and 150 colonies/plate) plates (see Table 4). Based on morphologies, selectivity is highest for babies' faeces with NPLN with 29% false positive colonies (growth, but no bifidobacterial morphology). PROP showed 39% false positive and RB with 50% false positives. False negatives (non-typical colonies, but bifid morphology) can be determined on RB, as this is the only medium for which typical colonies are described. However, no false negatives were observed in this work.


Legends on Table 4: b number in brackets is the percentage of false positive (typical colony, non-typical morphology) or false negatives (non-typical colony, typical morphology) of the colonies tested. c pos = colonies showing characteristics for bifidobacteria, neg = colonies not showing characteristics for bifidobacteria. Bifidobacteria characteristics were defined as yellow-green colonies with a yellow halo. This attribute could only be determined on RB, as no characteristics were defined for other media.

**Table 4.** Selectivity of media for bifidobacteria.

Most false positive colonies are reported to be different cocci (mono-, diplo- or streptococci), spore-forming rods and short rods. No yeast is observed on any of the media tested. Based on the actual counts, selectivity can only be determined for RB, as the colonies of bifidobacteria and non-bifidobacteria cannot be determined for the other media and not all colonies are tested for their morphology. Selectivity as percentages of non-typical colonies ranges from around 5 – 7%.

296 Lactic Acid Bacteria – R & D for Food, Health and Livestock Purposes

**Figure 6.** Counts (log CFU/gm wet weight) on PROP, NPLN and RB media in babies' faeces.

 **n typical non-typ. b**

RB pos c 24 12 12 (50)

RB neg 4 0 4 (0)

PROP 18 11 7 (39)

NPLN 28 20 8 (29)

Legends on Table 4: b number in brackets is the percentage of false positive (typical colony, non-typical morphology) or false negatives (non-typical colony, typical morphology) of the colonies tested. c pos = colonies showing characteristics for bifidobacteria, neg = colonies not showing characteristics for bifidobacteria. Bifidobacteria characteristics were defined as yellow-green colonies with a yellow halo. This attribute could only be determined on RB, as no

are described. However, no false negatives were observed in this work.

**Medium** 

0

2

4

6

**logN/gm**

8

10

12

characteristics were defined for other media.

**Table 4.** Selectivity of media for bifidobacteria.

Selectivity is also determined by microscopic observations of all different colony morphologies on all countable (between 10 and 150 colonies/plate) plates (see Table 4). Based on morphologies, selectivity is highest for babies' faeces with NPLN with 29% false positive colonies (growth, but no bifidobacterial morphology). PROP showed 39% false positive and RB with 50% false positives. False negatives (non-typical colonies, but bifid morphology) can be determined on RB, as this is the only medium for which typical colonies

PROP NPLN RB

**Babies' faeces**

**morphology**

123456 7 8 9 10 **Sample**

Colonies of different shapes can be tested microscopically. Bacterial morphology is determined, and typical and non-typical morphology is also determined. Typical morphology of bifidobacteria is branched or bifid-shaped rods. For the determination of bifidobacteria, none of the 3 media tested was decidedly selective. In this study, the occurrence of false positive or false negative colonies was determined. The lowest incidence of potential false positive colonies was observed on NPLN, but in all 3 media, the number of non-bifidobacteria capable of growing on the selective media was remarkably high. When many different species are capable of growing on the medium, an increase of one of these species may result in serious mistakes in calculating bifidobacteria. NPLN and RB gave slight higher counts than PROP. The incidence of false positive, based on morphologies on RB was comparable with that on the PROP and slightly higher than that on NPLN. The incidence of competitive flora was relatively low (less than 10% of the total colonies on the plates), as bifidobacteria are one of the main groups of intestinal bacteria in humans.

PROP medium has been described as the best medium for the determination of bifidobacteria by Silvi *et al*., (1996), but they also concluded that the total bifidobacterial counts were significantly lower on PROP than on the other media tested. Similarly, Favier *et al*., (1997) concluded that PROP underestimated bifidobacteria in some of their samples. Both studies used human faeces as the test substrate. Several other studies, in which PROP agar is used, also show significantly lower bifidobacterial counts than most other studies (Favier *et al*., 1997).

NPLN, which has been described as the medium of choice to choose bifidobacteria in dairy products, showed many cocci. This was in accordance with results observed by Silvi *et al*., (1996). In the same study, BIM-25 was tested, and this medium was found to be non-specific. All these 3 media performed reasonably well for human faeces and bifidobacteria can reliably be counted. The typical colonies morphological trait and the basic cellularmorphology of bifidobacteria were demonstrated well by RB media, with reference to NPNL and PROP medium. The RB medium presented strains with double thickness diameter and more bifurcated cellular morphology under phase contrast microscopy.
